LOCUS       sequence-243989-        6141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SPOON, a spontaneously blinking green 
            fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6141)
  AUTHORS   Arai Y, Takauchi H, Ogami Y, Fujiwara S, Nakano M, Matsuda T, Nagai 
            T
  TITLE     Spontaneously Blinking Fluorescent Protein for Simple Single Laser 
            Super-Resolution Live Cell Imaging.
  JOURNAL   ACS Chem Biol. 2018 Aug 17;13(8):1938-1943. doi: 
            10.1021/acschembio.8b00200. Epub 2018 Jul 10.
  PUBMED    29963852
REFERENCE   2  (bases 1 to 6141)
  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: 
            "10.1021/acschembio.8b00200"; journalName: "ACS Chem Biol"; date: 
            "2018-08-17- 17"; volume: "13"; issue: "8"; pages: "1938-1943"
COMMENT     Sequence Label: SPOON/ pcDNA3
FEATURES             Location/Qualifiers
     source          1..6141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     635..654
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        635..653
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(686..688,689..691,692..1402)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFVCTTGKLPVPWPSLLTTIGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNHDSHNVYITADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALGKDPNEKRDHMVLL
                     EFVTAAGITLGTDELYK"
     primer_bind     complement(731..752)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(992..1011)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(1466..1484)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1467..1484)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1504..1521)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1510..1734
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1780..2208
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1867..1886)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2077..2098
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2217..2237)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2222..2551
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2402..2537
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2464..2483
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2618..3412
                     /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(2672..2691)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3282..3301
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3586..3707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3623..3642)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3677..3696
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3756..3772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3756..3772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3769..3791)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3780..3796
                     /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(3804..3810,3811..3828,3829..3834))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3849..3870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3987..4004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4158..4746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4238..4257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4917..5708,5709..5777))
                     /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     5540..5559
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5778..5882)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5957..5976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      601 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      661 aagcttggta ccgagctcgg atccgatggt gagcaagggc gaggagctgt tcaccggggt
      721 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      781 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcgtct gcaccaccgg
      841 caagctgccc gtgccctggc cctccctcct gaccaccatc ggatacggcc tgatgtgctt
      901 cgcccgctat cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      961 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1021 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1081 ggaggacggc aacatcctgg ggcacaagct ggagtacaac cacgacagcc acaacgtcta
     1141 tatcacggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1201 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1261 ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctgg gcaaagaccc
     1321 caacgagaag cgcgatcaca tggtcctact ggagttcgtg accgccgccg ggatcaccct
     1381 cggcacggac gagctgtaca agtaagaatt ctgcagatat ccatcacact ggcggccgct
     1441 cgagcatgca tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat
     1501 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     1561 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     1621 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     1681 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     1741 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     1801 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1861 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1921 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1981 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2041 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2101 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2161 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     2221 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2281 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2341 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2401 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2461 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     2521 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     2581 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     2641 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     2701 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     2761 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     2821 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     2881 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     2941 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3001 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3061 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3121 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3181 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3241 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3301 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     3361 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     3421 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     3481 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3541 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3601 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3661 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3721 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3781 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3841 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3901 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3961 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4021 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4081 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4141 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4201 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4261 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4321 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4381 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4441 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4501 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4561 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4621 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4681 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4741 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4801 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4861 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4921 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4981 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5041 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5101 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5161 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5221 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5281 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5341 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5401 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5461 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5521 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5581 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5641 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5701 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5761 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5821 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5881 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5941 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6001 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     6061 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     6121 gcgatgtacg ggccagatat a
//
