LOCUS       sequence-142108-        4491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nuclear mCerulean2 (blue) fluorophore and a membrane targeted KOFP2 
            (orange) fluorophore separated by a self-cleaving 2A peptide for use
            with 5' promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4491)
  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 4491)
  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: pME-H2B-mCerulean3-P2A-mKOFP2-CAAX
FEATURES             Location/Qualifiers
     source          1..4491
                     /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    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1404..1423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1830..1886
                     /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             1893..2546
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     protein_bind    complement(2616..2715)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2732..2751)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2733..2751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2756..2772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2756..2772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2874..2893)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2885..3694
                     /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(2952..2971)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3841..4429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4330..4349
                     /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 tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctga attcgccgcc accatgcctg agcctagcaa gagcgcccct gcccctaaga
      721 agggaagcaa gaaggccatc accaaggccc agaagaagga cggaaagaag cgcaagagaa
      781 gccgcaaaga gtcttacagc atctacgtgt acaaggtgct gaaacaggtg caccctgaca
      841 ccggaatcag cagcaaggcc atgggaatca tgaacagctt cgtgaacgac atcttcgaga
      901 gaatcgccgg cgaggccagc agactggccc actacaacaa gagaagcacc atcaccagcc
      961 gcgagatcca gaccgccgtc agactgctgc tgcctggcga gctggccaag cacgccgtgt
     1021 ctgagggaac aaaggccgtg accaagtaca caagcagcaa gggcggagga agcggaggcg
     1081 gaagcggcgg aggatctatg gtgtctaagg gcgaggaact gttcaccggc gtggtgccta
     1141 tcctggtgga actggacggc gacgtgaacg gacacaagtt cagcgtgtcc ggcgagggcg
     1201 aaggcgacgc cacctacgga aagctgaccc tgaagttcat ctgcaccacc ggaaagctgc
     1261 ctgtgccttg gcctaccctg gtcaccaccc tgagctgggg agtgcagtgc ttcgccagat
     1321 accctgacca catgaagcag cacgatttct tcaagagcgc catgcctgag ggatacgtgc
     1381 aggaaagaac catcttcttc aaggacgacg gcaactacaa gaccagagcc gaagtgaagt
     1441 tcgagggcga caccctggtc aacagaatcg agctgaaggg aatcgacttc aaagaggacg
     1501 gaaacatcct gggacacaag ctggagtaca acgccatcca cggaaacgtg tacatcaccg
     1561 ccgacaagca gaagaacgga atcaaggcca acttcggcct gaactgcaac atcgaggacg
     1621 gaagcgtgca gctggccgac cactaccagc agaacacccc tatcggagat ggacctgtgc
     1681 tgctgccaga caaccactac ctgagcaccc agagcaagct gagcaaggac cctaacgaga
     1741 agcgcgacca catggtgctg ctggaatttg tgaccgccgc tggaatcacc ctgggaatgg
     1801 acgagctgta caagcctagg ggaagcggag ccaccaactt cagcctgctg aagcaggccg
     1861 gcgacgtgga agagaaccct ggacctcccg ggatggtgtc tgtgatcaag cctgagatga
     1921 agatgcgcta ctacatggac ggatctgtga acggccacga gtttaccatc gagggcgagg
     1981 gaaccggcag accttacgag ggacaccagg aaatgaccct gagagtcacc atggccgagg
     2041 gcggacctat gcctttcgcc ttcgacctgg tgtctcacgt gttctgctac ggacacagag
     2101 tgttcaccaa gtaccctgag gaaatccccg actacttcaa gcaggccttc cctgagggac
     2161 tgagctggga gagaagcctg gaatttgagg acggcggaag cgctagcgtg tccgcccaca
     2221 tcagcctgag aggaaacacc ttctaccaca agagcaagtt cacaggcgtg aacttccctg
     2281 ccgacggacc tatcatgcag aaccagagcg tggactggga gcctagcacc gagaagatca
     2341 ccgccagcga cggcgtgctg aagggcgacg tgaccatgta cctgaagctg gaaggcggag
     2401 gcaaccacaa gtgccagatg aagaccacct acaaggccgc caaagaaatc ctggaaatgc
     2461 caggcgacca ctacatcgga cacagactgg tccgcaagac cgagggaaac atcaccgagc
     2521 aggtcgagga cgccgtggct cacagcaagc tgaaccctcc agacgagagc ggacctggat
     2581 gcatgagctg caaatgcgtg ctgagctgaa ctagtaccca gctttcttgt acaaagttgg
     2641 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     2701 taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat
     2761 agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     2821 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     2881 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     2941 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     3001 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     3061 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     3121 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     3181 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     3241 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     3301 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     3361 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     3421 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     3481 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     3541 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     3601 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     3661 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     3721 ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     3781 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3841 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3901 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3961 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4021 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4081 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4141 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4201 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4261 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4321 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4381 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4441 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
