LOCUS       sequence-386837-        7911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  zebrafish expression of nbt-mTagBFP2-P2A-mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7911)
  AUTHORS   Papadaki S, Wang X, Wang Y, Zhang H, Jia S, Liu S, Yang M, Zhang D, 
            Jia JM, Koster RW, Namikawa K, Piatkevich KD
  TITLE     Dual-expression system for blue fluorescent protein optimization.
  JOURNAL   Sci Rep. 2022 Jun 17;12(1):10190. doi: 10.1038/s41598-022-13214-0.
  PUBMED    35715437
REFERENCE   2  (bases 1 to 7911)
  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: "2022-06-17"; volume: "12(1):10190. doi"; pages: " 
            10.1038/s41598-022-13214-0"
COMMENT     Sequence Label: pTol2-nbt-mTagBFP2-P2A-mScarlet
FEATURES             Location/Qualifiers
     source          1..7911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(30..41,42..179)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        187..205
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3738..3747
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3744..4454
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             4473..4529
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4530..5225
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(4663..4681)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4931..4950
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5271..5405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5295..5314)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5349..5368
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(5508..5526)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(5533..5720,5721..5732)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5762..5778)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5762..5778)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5775..5797)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5786..5802
                     /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(5810..5816,5817..5834,5835..5840))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5855..5876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5993..6010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6164..6752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6244..6263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6923..7714,7715..7783))
                     /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     7546..7565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7784..7888)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaatc ccatatgggc agaggtgtaa aaagtactca aaaattttac
       61 tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa aagtatctgg
      121 ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac ttgagtatta
      181 gatctgtaat acgactcact atagggacat agaatgcagg tacgtacccc tcgaggtcga
      241 ctctagaccc tgtctgttcc tgattacatc tagtttgctg cctgtccgga ttattgcctg
      301 ttttttgact actctcttgt atcctgtggt gtactttcta ttttggtgtg tttgactcgg
      361 cctgtgaccc caaccattta tttgtctcct gtaactgtac cgcattgact gattgttact
      421 gacccggcct ggcttttaac tacaattctt gtttccaatt ctacaacccc acacttggtt
      481 cccttgcttg gtcagaactc tctccttggg cctctcacgt taagacctgg cgtcatctga
      541 gtagcagagg gctagtcccg atgccaaagg cggctgttat aggcagaagg gtgagccgcg
      601 actgggtcct tggtgtctgt tctgattttg ggacaccctg ccttacagcc cccctcaagt
      661 ctctgattat tgccttgtaa ccttgtaacc aatcagtgga aatcaagaga cctgcaaagc
      721 aggaagtagt gttctactta tgtcactcca gtctttatac attacatttt tggctaacta
      781 actatattag aaacattttt tattttgcat agccaatcta cagtatttac ccagttttta
      841 tttttaaact gaataattat tttaaggata atatctgcct gcagaataaa taattcgata
      901 tatgccatgg ataagcaata tggtgtcttg acaagacaga atataatttg atattttaga
      961 agcatggctt ttatggactt atacacatgt aggtatgatg tgagaacctg gttaatggtg
     1021 tccatttgct gtcatgagtt tgcttggtta ctgagcacag actattttat gagctttgcc
     1081 aggtcctgct tttctcatcc tttcaaaaca atgtgttccc ttcactccac acagagtaat
     1141 gtccatgttc ctggtgtgtg agtctcctct tccaaatgca gcatcttctc cccccccatg
     1201 ccatgtgaca ctttcattag catctcctca tgctttgtgt gaattctgct ctgccctacc
     1261 cctcccctta tgcagcacaa ggaagaaggg agaaagctgt gtctgcatat ctgcaaaagg
     1321 ctgcctgact gacagtaaga atgtctgtct cgaggacctc ttcccacaca gctgtaccac
     1381 tgaaatccat atttattggt agtacatctg ttactaacat tttaacatta acaacagtga
     1441 ttacattacc ttcagaacat tgcagtaagg aacagactcc tgtatgctca tagtgtaata
     1501 ccaggagaat atattgtaaa tcttgggtgt tgcccacttt cagtgatatc agctttgctt
     1561 tctgtatttt ttaatcattt aatgctcctt catagtaatc ttgaaatgca aacattttcc
     1621 ttaaggtggg catagccaaa caatattcaa catcaacaca ctgggccaaa tcaggatgat
     1681 ctgatcaaca ggctaacaat cagatcatat ttgggagcca tttgccaatg tggtcctctt
     1741 ccaacatgat tatccaaact gtctgataga tatatctgct tgatagtcaa atattggtca
     1801 ggcaggccat tgagaaggcc tcatgcatgg gccaataagc tgctgattgg gttgcctact
     1861 tgtggatatt ttactagcct ggccagaaaa aatgttgctt gatgccaatg tcaataattg
     1921 gaaaaaaaca ttaaaatata agaaggctgt tacttttttc ccagaaaagg tggtaaccat
     1981 aactgccaac taattctgga agagctgagt tggcagctta tacttggcca tttatcaatc
     2041 accacctata aggatgtacc gagtctgcgt gcttattagg tatatgtgag tatgacacaa
     2101 ttttaaacac tattatgtct gcaagagctg agtcagcagt tatacttggc catgtattgc
     2161 cacccataag gatgtactga ttcaatcatt tttctccaaa cccctaaatt catatgatgt
     2221 ctgccagctt attaggtaca gtatatgtga gtatgataaa aatatttgta atatttaata
     2281 ttggttgtgt tttctctcta tagaaataat acacaatcca aacaaaagtg gcataaatgt
     2341 gatatattgt aatgacatga taaggaaatt acactgattt attggctatt aatatttctc
     2401 atttgcaatc actatgctgc tctggcagct tttatcagca catcagtgct acagaaagaa
     2461 tggaaccctg gctgtcatgc tgattctaat gctcagcagc attttacaca ttttgctgca
     2521 ttttctagct atcagctgat tactcccagg atcccccttg tgttccactg cagatgtatt
     2581 tactgatgct tagcttctac caacgggcac ttacaagtat ttctatgtgg gtgtaactag
     2641 aaagaagatg gactatgtaa agcatatggg cctctagttt gggcataacc ttaatgtaca
     2701 tttggatggg aggttccagg gtaaaggtta atccaatagc actggtttcc cctagctttg
     2761 ccacttttaa taggcaaggt ttcagccctg tctactcata tgtaaattgt gattgtgatt
     2821 ttatttcaca tttttgttct gtatgtttgg acagatgtca gtaaaaccat ttaactgccc
     2881 ccgtttgttc aacctcctcg ttcttctgct acatccactg gtctttacgg aagcaattaa
     2941 ataaattaag ggccatatga tttcctgccc tgtaaaactg gtgtgtgttt gcggtctgag
     3001 gcagatgtgc tgcttccatt tgaatacagg ggggatattg aaacactgac tcgggaatcc
     3061 cctggagagt gatttgaggc agaaatggat atggagcagt atcatgtgat aaaatgaaat
     3121 tgtgaggcag ctgcataggg tggcacccat ccccactgca ccttccttcc ccctacagct
     3181 cccagtgatg gacgagttgt gtgggggatg gtacctgtca ctaatacctt tggagtcatc
     3241 attaggctaa tattatcaat aacaaggtta atgtttctat aggcttaatc agcttctcat
     3301 tcgtgtttta attagaattt tttttcatta tttcagtata atattccttt gtaataagca
     3361 ataagcacat tcccagcatc tttttcacct tcttggtggt atctgcctga tggtgagaca
     3421 atgcagcatc catttccatt ccctcccttg tcatttgatt ggttcccatc cctacaatgc
     3481 tcagggccag tggctccacc caccattaca tcatctgagc ccaggaccac agcaagtgaa
     3541 taggagagag cagcaccctc cccaaggcgt cacaagtgta taaaaggcag cagaaggagc
     3601 ctctgacagc ccagattctt agccggctgg aaaccatcgg ggtgttgcac aaagggcaag
     3661 taaagcaaca agaaagggcc acaccatcca ctgattacac cactgtaaga ctcaacccaa
     3721 tcaagcttga attcgccgcc accatggtgt ctaagggcga agagctgatt aaggagaaca
     3781 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     3841 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     3901 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     3961 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     4021 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     4081 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     4141 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     4201 ccgctgacgg cggactggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     4261 atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag aacctcaaga
     4321 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     4381 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     4441 ggcacaagct gaatgggcgc gccggaagcg gagctactaa cttcagcctg ttaaagcaag
     4501 caggagacgt cgaggagaac cctggaccta tggtgagcaa gggcgaggca gtgatcaagg
     4561 agttcatgcg gttcaaggtg cacatggagg gctccatgaa cggccacgag ttcgagatcg
     4621 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     4681 agggtggccc cctgcccttc tcctgggaca tcctgtcccc tcagttcatg tacggctcca
     4741 gggccttcac caagcacccc gccgacatcc ccgactacta taagcagtcc ttccccgagg
     4801 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc gtgacccagg
     4861 acacctccct ggaggacggc accctgatct acaaggtgaa gctccgcggc accaacttcc
     4921 ctcctgacgg ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc accgagcggt
     4981 tgtaccccga ggacggcgtg ctgaagggcg acattaagat ggccctgcgc ctgaaggacg
     5041 gcggccgata cctggcggac ttcaagacca cctacaaggc caagaagccc gtgcagatgc
     5101 ccggcgccta caacgtggac cgcaagttgg acatcacctc ccacaacgag gactacaccg
     5161 tggtggaaca gtacgaacgc tccgagggcc gccactccac cggcggcatg gacgagctgt
     5221 acaagtaaat ctagtctcga ggctagaact atagtgagtc gtattacgta gatccagaca
     5281 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     5341 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     5401 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     5461 ttttttaatt cgcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     5521 ttaattgggc ccaagtgatc tccaaaaaat aagtactttt tgactgtaaa taaaattgta
     5581 aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta aaagtattga
     5641 tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag tacagtaatc
     5701 aagtaaaatt actcaagtac tttacacctc tgggtactgt ttcgcgcttg gcgtaatcat
     5761 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5821 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5881 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5941 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6001 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6061 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6601 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6781 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6841 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6901 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7021 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7081 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7201 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7741 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7861 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
