LOCUS       sequence-145999-        4568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear mCerulean3 (blue) fluorophore and a membrane targeted KOFP2 
            (orange) fluorophore separated by a self-cleaving 2A peptide for use
            with C-terminal fusions..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4568)
  AUTHORS   Don EK, Formella I, Badrock AP, Hall TE, Morsch M, Hortle E, Hogan 
            A, Chow S, Gwee SS, Stoddart JJ, Nicholson G, Chung R, Cole NJ
  TITLE     A Tol2 Gateway-Compatible Toolbox for the Study of the Nervous 
            System and Neurodegenerative Disease.
  JOURNAL   Zebrafish. 2016 Sep 15.
  PUBMED    27631880
REFERENCE   2  (bases 1 to 4568)
  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: "Zebrafish. 
            2016 Sep 15."
COMMENT     Sequence Label: p3E-H2B-mCerulean3-P2A-mKOFP2-CAAX
FEATURES             Location/Qualifiers
     source          1..4568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1465..1484)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1891..1947
                     /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             1954..2607
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     protein_bind    complement(2677..2772)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2809..2828)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2810..2828)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2833..2849)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2833..2849)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2951..2970)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2962..3771
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3029..3048)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3918..4506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4407..4426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtgggaa ttcgccgcca ccatgcctga accctctaag
      781 aagggaagca agaaggccat caccaaggcc cagaagaagg acggaaagaa gcgcaagaga
      841 agccgcaaag agtcttacag catctacgtg tacaaggtgc tgaaacaggt gcaccctgac
      901 accggaatca gcagcaaggc catgggaatc atgaacagct tcgtgaacga catcttcgag
      961 agaatcgccg gcgaggccag cagactggcc cactacaaca agagaagcac catcaccagc
     1021 cgcgagatcc agaccgccgt cagactgctg ctgcctggcg agctggccaa gcacgccgtg
     1081 tctgagggaa caaaggccgt gaccaagtac acaagcagca agggcggagg aagcggaggc
     1141 ggaagcggcg gaggatctat ggtgtctaag ggcgaggaac tgttcaccgg cgtggtgcct
     1201 atcctggtgg aactggacgg cgacgtgaac ggacacaagt tcagcgtgtc cggcgagggc
     1261 gaaggcgacg ccacctacgg aaagctgacc ctgaagttca tctgcaccac cggaaagctg
     1321 cctgtgcctt ggcctaccct ggtcaccacc ctgagctggg gagtgcagtg cttcgccaga
     1381 taccctgacc acatgaagca gcacgatttc ttcaagagcg ccatgcctga gggatacgtg
     1441 caggaaagaa ccatcttctt caaggacgac ggcaactaca agaccagagc cgaagtgaag
     1501 ttcgagggcg acaccctggt caacagaatc gagctgaagg gaatcgactt caaagaggac
     1561 ggaaacatcc tgggacacaa gctggagtac aacgccatcc acggaaacgt gtacatcacc
     1621 gccgacaagc agaagaacgg aatcaaggcc aacttcggcc tgaactgcaa catcgaggac
     1681 ggaagcgtgc agctggccga ccactaccag cagaacaccc ctatcggaga tggacctgtg
     1741 ctgctgccag acaaccacta cctgagcacc cagagcaagc tgagcaagga ccctaacgag
     1801 aagcgcgacc acatggtgct gctggaattt gtgaccgccg ctggaatcac cctgggcatg
     1861 gatgaactgt ataaacctag gggaagcgga gccaccaact tcagcctgct gaagcaggcc
     1921 ggcgacgtgg aagagaaccc tggacctccc gggatggtgt ctgtgatcaa gcctgagatg
     1981 aagatgcgct actacatgga cggatctgtg aacggccacg agtttaccat cgagggcgag
     2041 ggaaccggca gaccttacga gggacaccag gaaatgaccc tgagagtcac catggccgag
     2101 ggcggaccta tgcctttcgc cttcgacctg gtgtctcacg tgttctgcta cggacacaga
     2161 gtgttcacca agtaccctga ggaaatcccc gactacttca agcaggcctt ccctgaggga
     2221 ctgagctggg agagaagcct ggaatttgag gacggcggaa gcgctagcgt gtccgcccac
     2281 atcagcctga gaggaaacac cttctaccac aagagcaagt tcacaggcgt gaacttccct
     2341 gccgacggac ctatcatgca gaaccagagc gtggactggg agcctagcac cgagaagatc
     2401 accgccagcg acggcgtgct gaagggcgac gtgaccatgt acctgaagct ggaaggcgga
     2461 ggcaaccaca agtgccagat gaagaccacc tacaaggccg ccaaagaaat cctggaaatg
     2521 ccaggcgacc actacatcgg acacagactg gtccgcaaga ccgagggaaa catcaccgag
     2581 caggtcgagg acgccgtggc tcacagcaag ctgaaccctc cagacgagag cggacctgga
     2641 tgcatgagct gcaaatgcgt gctgagctga actagtcaac tttattatac aaagttggca
     2701 ttataaaaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata
     2761 aaatcattat ttggagctcc atggtagcgt taacgcggcc gcgatatccc ctatagtgag
     2821 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     2881 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     2941 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     3001 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     3061 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     3121 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     3181 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     3241 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     3301 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     3361 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     3421 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     3481 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     3541 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     3601 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     3661 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     3721 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     3781 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
     3841 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     3901 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3961 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4021 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4081 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4141 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4201 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4261 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc
     4321 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4381 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4441 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4501 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4561 cacatgtt
//
