LOCUS       sequence-380348-        8402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  zebrafish expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8402)
  TITLE     Koster Lab Plasmids 2022
REFERENCE   2  (bases 1 to 8402)
  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"
COMMENT     Sequence Label: pTol2-nbt300
            2n-KalNFB-3xmir1T-syt2a-10U2n-mCa...
FEATURES             Location/Qualifiers
     source          1..8402
                     /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        188..206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             626..1066
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             1514..1885
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     polyA_signal    3351..3432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3581..3715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3846..3940
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     protein_bind    4029..4123
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     regulatory      4199..4208
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4205..4936
                     /codon_start=1
                     /product="far-red fluorescent protein derived from 
                     mNeptune, with further red-shifted spectra"
                     /label=mCardinal
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTTEGEGKPYEGTQTQ
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEATTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             4955..5011
                     /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             join(5012..5014,5015..5017,5018..5731)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(5057..5078)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     5665..5686
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5762..5896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(5999..6017)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(6024..6211,6212..6223)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6253..6269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6253..6269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6266..6288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6277..6293
                     /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(6301..6307,6308..6325,6326..6331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6346..6367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6484..6501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6655..7243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6735..6754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7414..8205,8206..8274))
                     /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     8037..8056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8275..8379)
                     /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 gatcctgtaa tacgactcac tataggttaa ttaagacaaa gaatgcaggt acacgagcag
      241 catccatttc cattccctcc cttgtcattt gattggttcc catccctaca atgctcaggg
      301 ccagtggctc cacccaccat tacatcatct gagcccagga ccacagcaag tgaataggag
      361 agagcagcac cctccccaag gcgtcacaag tgtataaaag gcagcagaag gagcctctga
      421 cagcccagat tcttagccgg ctggaaacca tcggggtgtt gcacaaaggg caagtaaagc
      481 aacaagaaag ggccacacca tccactgatt acaccactgt aagactcaac ccaatcaagc
      541 ttggatccct tcagcaccaa ggacagggcc tctaggacac tagaggtttc agcaccaagg
      601 acagggcctc gaattcgccg ccaccatgaa actgctctca tccatcgagc aagcctgcga
      661 catttgtcgg cttaagaagc tgaaatgctc caaggaaaag ccgaaatgtg ccaaatgcct
      721 gaagaacaat tgggaatgtc gttactctcc caaaaccaag cgaagtccac tcacaagggc
      781 tcatctgacc gaagtggaga gcaggctaga gagactggaa caactctttt tgctcatctt
      841 ccctagagag gaccttgaca tgatcctcaa gatggattct ctccaggata ttaaagccct
      901 tttgactggc ttattcgtcc aggacaatgt gaacaaagac gctgtgacag accgattggc
      961 aagtgtcgag accgatatgc ctctgacact gagacagcac agaatcagcg ctacttcctc
     1021 aagcgaagag tcttctaaca agggacagag acagctgact gtttcgagca ggtcgaggcc
     1081 catggagttc cagtacctgc cagatacaga cgatcgtcac cggattgagg agaaacgtaa
     1141 aaggacatat gagaccttca agagcatcat gaagaagagt cctttcagcg gacccaccga
     1201 cccccggcct ccacctcgac gcattgctgt gccttcccgc agctcagctt ctgtccccaa
     1261 gccagcaccc cagccctatc cctttacgtc atccctgagc accatcaact atgatgagtt
     1321 tcccaccatg gtgtttcctt ctgggcagat cagccaggcc tcggccttgg ccccggcccc
     1381 tccccaagtc ctgccccagg ctccagcccc tgcccctgct ccagccatgg tatcagctct
     1441 ggcccaggcc ccagcccctg tcccagtcct agccccaggc cctcctcagg ctgtggcccc
     1501 acctgccccc aagcccaccc aggctgggga aggaacgctg tcagaggccc tgctgcagct
     1561 gcagtttgat gatgaagacc tgggggcctt gcttggcaac agcacagacc cagctgtgtt
     1621 cacagacctg gcatccgtcg acaactccga gtttcagcag ctgctgaacc agggcatacc
     1681 tgtggccccc cacacaactg agcccatgct gatggagtac cctgaggcta taactcgcct
     1741 agtgacaggg gcccagaggc cccccgaccc agctcctgct ccactggggg ccccggggct
     1801 ccccaatggc ctcctttcag gagatgaaga cttctcctcc attgcggaca tggacttctc
     1861 agccctgctg agtcagatca gctcctaatc tagccctagg tgtacaggcg cgccactagt
     1921 tctacctctc gagaatctag aatacatact tctaatcatt ccatagctaa tacatacttc
     1981 taatcattcc atagctaata catacttcta atcattccat actagaacta tagtgagtcg
     2041 tagatctcgc gccaacccgc tcctcctccc tccctccctc cctccctcct ctcctttatt
     2101 cacttttcaa catgcacaac acgtcatcaa atcccccaat cagcactgaa ttacagccac
     2161 gggccaccag actgaaggtg ttaaagaaga cgattcggtc tcatcagcgg ctaaaacgag
     2221 atgagggtga accgagagag actgtttcca cagatgttct tgaaggaggg ccgggctcgt
     2281 aggtgcggtt attacacata taagctccca ttcacacctt aaaacatcca acagcattcc
     2341 aaagttttcc atcggggacc agggatagaa aacactgcct cttccactgc ctaatttaac
     2401 acattccttc tagattatct ctggtcatct agatgaaatc agttctgttc tgtttgccgt
     2461 gtctgaaaag acagtaaaga gtaattgaaa aaaaaaatga atatagaaaa atggggactg
     2521 gagagatatt tggactagac gctcctgatc cagccagaag gaacctttca ggtgtcgatt
     2581 acactgcctg cgaaaacaac ctcttcttcc attcttcagt ttccagctgc cggctggctt
     2641 cttccactgc ccaacatcct aacaaacccc tgacaaaaaa ggagacaagc cttgaaacat
     2701 cctgaaataa gcaattctta gctgccaggg acgtcttcct gttagcatca gtgtatttgt
     2761 ttcttttgct ttgatttgtt ttcttttttt attttatttt catggtaaat gtattctgcc
     2821 tgagcacttt aaacgaaggt gtttttgttc cagtcgatgc ttcgttttga acctgctttt
     2881 atatttatta acttttgttg tgtattttta atgtttaatc tttaaaggaa tagttcacct
     2941 acacacacac acacacacac acacacacac acaaatgaaa tgaagtcatc atttactcac
     3001 ttttctttct tttagctgtg gagcacaaaa cgagatgttt tgaagaatgt tcacgctgtt
     3061 gtttttcatg caatttaagt aaacgaagat caagggatgt tgagctatac aaatattcat
     3121 tatatgggca gcacggtcgc tggttcgagt cccggctggg ccagttggca tttctgagtt
     3181 tgcatattct ccctgtgttg gcatgggttt cctcctggtg ctccgtttct cccacagtcc
     3241 actagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     3301 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     3361 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3421 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatca ggccttggta
     3481 ccgggcccaa tgcattggcg ccgcggccgg ccgcgaatta aaaaacctcc cacacctccc
     3541 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3601 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3661 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctacgt
     3721 aatacgactc actatagttc tagcctcgag actagatcgt ttaaacagat cgtttaaacg
     3781 tgtggaggag ctcaaagtga ggctgagacg cgatgggatc cattatatac cctctagagt
     3841 ctccgctcgg aggacagtac tccgctcgga ggacagtact ccgctcggag gacagtactc
     3901 cgctcggagg acagtactcc gctcggagga cagtactccg acctgcaatt taaattcagc
     3961 accaaggaca gggcctctag gacactagag gtttcagcac caaggacagg gccttaatta
     4021 atgcaggtcg gagtactgtc ctccgagcgg agtactgtcc tccgagcgga gtactgtcct
     4081 ccgagcggag tactgtcctc cgagcggagt actgtcctcc gagcggagac tctagagggt
     4141 atataatgga tcccatcgcg tctcagcctc actttgagct cctccacacg aattcgccgc
     4201 caccatggtg agcaagggcg aggagctgat caaggagaac atgcacatga agctgtacat
     4261 ggaaggcacc gtgaacaacc accacttcaa gtgcaccacc gaaggggagg gcaagcccta
     4321 cgagggcacc cagacccaga ggattaaggt ggtggaggga ggccccctgc cgttcgcatt
     4381 cgacatcctg gccacctgct ttatgtacgg gagcaagacc ttcatcaacc acacccaggg
     4441 catccccgat ttctttaagc agtccttccc tgagggcttc acatgggaga gagtcaccac
     4501 atacgaagac gggggcgtgc ttaccgttac ccaggacacc agcctccagg acggctgctt
     4561 gatctacaac gtcaagctca gaggggtgaa cttcccatcc aacggccctg tgatgcagaa
     4621 gaaaacactc ggctgggagg ccaccaccga gaccctgtac cccgctgacg gcggcctgga
     4681 aggcagatgc gacatggccc tgaagctcgt gggcgggggc cacctgcact gcaacctgaa
     4741 gaccacatac agatccaaga aacccgctaa gaacctcaag atgcccggcg tctactttgt
     4801 ggaccgcaga ctggaaagaa tcaaggaggc cgacaatgag acctacgtcg agcagcacga
     4861 ggtggctgtg gccagatact gcgacctccc tagcaaactg gggcacaaac ttaatggcat
     4921 ggacgagctg tacaaggggc gcgccggaag cggagctact aacttcagcc tgttaaagca
     4981 agcaggagac gtcgaggaga accctggacc tatggtgagc aagggcgagg agctgttcac
     5041 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     5101 ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
     5161 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcgtggc
     5221 ctgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     5281 cgaaggctac gtccaggagc gcaccatctc tttcaaggac gacggcacct acaagacccg
     5341 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     5401 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacta
     5461 cgtctatatc acggccgaca agcagaagaa ctgcatcaag gctaacttca agatccgcca
     5521 caacgttgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     5581 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc catcagtcca agctgagcaa
     5641 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     5701 tacacatggc atggacgagc tgtacaagta aatctagaac tatagtgagt cgtattacgt
     5761 agatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     5821 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     5881 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     5941 ggtgtgggag gttttttaat tcgcggccgc caccgcggtg gagctccagc ttttgttccc
     6001 tttagtgagg gttaattggg cccaagtgat ctccaaaaaa taagtacttt ttgactgtaa
     6061 ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt
     6121 aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa
     6181 gtacagtaat caagtaaaat tactcaagta ctttacacct ctgggtactg tttcgcgctt
     6241 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     6301 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     6361 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6421 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6481 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6541 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6601 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6661 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6721 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6781 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6841 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6901 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6961 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7021 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7081 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7141 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7201 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7261 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7321 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7381 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7441 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7501 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7561 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7621 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7681 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7741 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7801 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7861 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7921 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7981 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     8041 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8101 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8161 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8221 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8281 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8341 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8401 ac
//
