LOCUS       sequence-386839-        8386 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 8386)
  TITLE     Koster Lab Plasmids 2022
REFERENCE   2  (bases 1 to 8386)
  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..8386
                     /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    4013..4107
                     /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      4183..4192
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4189..4920
                     /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             4939..4995
                     /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(4996..4998,4999..5001,5002..5715)
                     /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(5041..5062)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     5649..5670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5746..5880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(5983..6001)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(6008..6195,6196..6207)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6237..6253)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6237..6253)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6250..6272)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6261..6277
                     /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(6285..6291,6292..6309,6310..6315))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6330..6351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6468..6485)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6639..7227)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6719..6738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7398..8189,8190..8258))
                     /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     8021..8040
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8259..8363)
                     /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 actaatcgtt taaacagatc agtttaacgt
     3781 gtggaggagc tcaaagtgag gctgagacgc gatgggatcc attatatacc ctctagagtc
     3841 tccgctcgga ggacagtact ccgacctgca gcccggggga tcgtcgacag gtcctagcta
     3901 gggctgctct ccaatctctg tcatggcagg gccactggca tccctgaagt tggtggagac
     3961 cacctcactc cactcagcat acagctcatc cagtcctctt agggctgcag gtcggagtac
     4021 tgtcctccga gcggagtact gtcctccgag cggagtactg tcctccgagc ggagtactgt
     4081 cctccgagcg gagtactgtc ctccgagcgg agactctaga gggtatataa tggatcccat
     4141 cgcgtctcag cctcactttg agctcctcca cacgaattcg ccgccaccat ggtgagcaag
     4201 ggcgaggagc tgatcaagga gaacatgcac atgaagctgt acatggaagg caccgtgaac
     4261 aaccaccact tcaagtgcac caccgaaggg gagggcaagc cctacgaggg cacccagacc
     4321 cagaggatta aggtggtgga gggaggcccc ctgccgttcg cattcgacat cctggccacc
     4381 tgctttatgt acgggagcaa gaccttcatc aaccacaccc agggcatccc cgatttcttt
     4441 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     4501 gtgcttaccg ttacccagga caccagcctc caggacggct gcttgatcta caacgtcaag
     4561 ctcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg
     4621 gaggccacca ccgagaccct gtaccccgct gacggcggcc tggaaggcag atgcgacatg
     4681 gccctgaagc tcgtgggcgg gggccacctg cactgcaacc tgaagaccac atacagatcc
     4741 aagaaacccg ctaagaacct caagatgccc ggcgtctact ttgtggaccg cagactggaa
     4801 agaatcaagg aggccgacaa tgagacctac gtcgagcagc acgaggtggc tgtggccaga
     4861 tactgcgacc tccctagcaa actggggcac aaacttaatg gcatggacga gctgtacaag
     4921 gggcgcgccg gaagcggagc tactaacttc agcctgttaa agcaagcagg agacgtcgag
     4981 gagaaccctg gacctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     5041 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag
     5101 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     5161 gtgccctggc ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac
     5221 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     5281 gagcgcacca tctctttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc
     5341 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     5401 aacatcctgg ggcacaagct ggagtacaac ttcaacagcc actacgtcta tatcacggcc
     5461 gacaagcaga agaactgcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc
     5521 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     5581 ctgcccgaca accactacct gagccatcag tccaagctga gcaaagaccc caacgagaag
     5641 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac
     5701 gagctgtaca agtaaatcta gaactatagt gagtcgtatt acgtagatcc agacatgata
     5761 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     5821 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     5881 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     5941 taattcgcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat
     6001 tgggcccaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     6061 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     6121 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     6181 aaattactca agtactttac acctctgggt actgtttcgc gcttggcgta atcatggtca
     6241 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6301 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6361 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6421 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6481 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6541 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6601 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6661 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6721 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6781 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6841 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6901 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6961 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7021 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     7081 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7141 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7201 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7261 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7321 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7381 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7441 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7501 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7561 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7621 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7681 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7741 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7801 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7861 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7921 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7981 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8041 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8101 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8161 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8221 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8281 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8341 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
