LOCUS       sequence-380747-        7311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for conditional, intronic, targeted integration of a 
            genebreak cassette optimized for zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7311)
  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: GBH-2aG4-gcryBFP.3
FEATURES             Location/Qualifiers
     source          1..7311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(17..28,29..191)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    278..299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(314..319,320..337,338..344)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    352..368
                     /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)."
     primer_bind     357..379
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     376..392
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     376..392
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             388..738
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSLLDNGPI
                     TLNNANYLKLLS"
     promoter        413..431
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(479..497)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(507..524)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(507..523)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(516..538)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    698..729
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(735..768)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             944..1000
                     /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             1007..1444
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3530..3550
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3554..4255
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4265..4489
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4559..4640
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4764..4845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4994..5027)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5033..5064
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5069..5102
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5282..5300
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5323..5510,5511..5522)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5576..5595)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5587..6396
                     /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(5654..5673)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6539..7127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7028..7047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7281..7298
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttaaatggt accgagcaga ggtgtaaaaa gtactcaaaa attttactca agtgaaagta
       61 caagtactta gggaaaattt tactcaatta aaagtaaaag tatctggcta gaatcttact
      121 tgagtaaaag taaaaaagta ctccattaaa attgtacttg agtattaagg aagtaaaagt
      181 aaaagcaaga aagaaaacta gagtggtctc cctttagtga gggtcaattg atgggaggcg
      241 ttcgggccac agcggacacg caggtgcgca acgcaattaa tgtgagttag ctcactcatt
      301 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      361 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
      421 ctcactaaag ggaacaaaag ctggaggacc tcgagggggg gcccggtacc caattcgccc
      481 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
      541 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
      601 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
      661 tggacctgcc acacttcctt gctagacaat gggcccataa ctttaaataa tgccaattat
      721 ttaaagttac tatcataact tcgtatagca tacattatac gaagttatac ctaggcttac
      781 cacccactag cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt
      841 cacatgctca ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct
      901 cattcattta cagcagccgg aattcacccg ggtaccggaa ggtgccacga acttctctct
      961 gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct tctagaaagc tactgtcttc
     1021 tatcgaacaa gcatgcgata tttgccgact taaaaagctc aagtgctcca aagaaaaacc
     1081 gaagtgcgcc aagtgtctga agaacaactg ggagtgtcgc tactctccca aaaccaaaag
     1141 gtctccgctg actagggcac atctgacaga agtggaatca aggctagaaa gactggaaca
     1201 gctatttcta ctgatttttc ctcgagaaga ccttgacatg attttgaaaa tggattcttt
     1261 acaggatata aaagcattgt taacaggatt atttgtacaa gataatgtga ataaagatgc
     1321 cgtcacagat agattggctt cagtggagac tgatatgcct ctaacattga gacagcatag
     1381 aataagtgcg acatcatcat cggaagagag tagtaacaaa ggtcaaagac agttgactgt
     1441 atcgtcgagg tcgaccccgg gaatacagat ctctcgagcc gcccccccga ccgatgtcag
     1501 cctgggggac gagctccact tagacggcga ggacgtggcg atggcgcatg ccgacgcgct
     1561 agacgatttc gatctggaca tgttggggga cggggattcc ccgggtccgg gatttacccc
     1621 ccacgactcc gccccctacg gcgctctgga tatgtaacta gtaattaagt ctcagccacc
     1681 gttaactgaa catgtcaaaa cctgtggaga ctgttgagat ttgatgttct gaaaagataa
     1741 agcctataaa taaaatgttg cccaaatttc ctgcctgatg tttttctttg tctttgctac
     1801 atggctttgc tgctcggatc ggctcactct gtgtatgcca cgttcacttt gtactctcct
     1861 tctcacggta ggtttattat ttttagatgt gcagttagtt tctgtgaaat aacacaccac
     1921 acactgatat tgtctgtgca ttgacttggt gagtgcacat tgtttttgat cttgacatat
     1981 ttatatttga ttgatcaggt gaactgtgtg aatctaaagt gctccataca gatgttctgc
     2041 attgaaaata ttctcatttt attagtggaa gtgagtgtat gccacatcca atcaatttca
     2101 gcaaacaccc cagtatgatt taatgcaaaa aaatgaaggt atcaaacacg cattactact
     2161 ttgcagttaa atatttaaca tttattccaa cacgaaaaaa agcagtaaat aacactttga
     2221 caaacacgtc aggacatctt atttttgtca ccctcacagg caatttagta taatatatta
     2281 tatatatata tatatcatat aataatattc agtataatat atatatatat atcatattat
     2341 aatattcagt ataatataaa acacaaacac atatatgtat aatataatat aacattttta
     2401 tttattgaga tgcctctatg gaccgtgtta taagaagtaa agatcaggag aagtaaacat
     2461 gaagtgtaat tatgaatact gatgttaaat taagctatga tgagttttca ctgttaattt
     2521 accatctcaa ttaaatgttg atgcctccat gaccaagtta agcagatgag actgaggaca
     2581 actgtagaag acaagatgtt cactttgctg aatatagctg gcttgacagt tatctatgac
     2641 tctataaata tatatatatt ttttttttta taaaatgatt tatttataac tatatatcca
     2701 tttctcagac aggtgcttca tatccctcac tcccgtagct gtccatgctg gatctgtccc
     2761 cgttgttttt aaaaagctaa ataagttatt aacatgactg catccagcga gccaaacctg
     2821 tctggtgtac agctaccaga gaagcttgag atcctagtca ccggcgttcc tagttcttta
     2881 aactgttaca aagtgtttgg accagcaact gaattgcact gtaaattacc acatcagggt
     2941 ctccggggtc aacgctgcta agcaatggac tggaaaattt gtgacattga gtttttggaa
     3001 taactgggaa tgctggataa ctctttgggt aaagctatgg aacaaaagta ccagcatgaa
     3061 ctaagaaatg actgtcacag cacaacatca aacacaatgc aagacaaagg ttctctgctt
     3121 ttgccttact tgacagaaaa agtaagctct aggttcatcg catgcagaca gcagcaatac
     3181 atgacatata taaaggctgg ctcttttctc ccttccattg aactgaaact tccactcagt
     3241 cagacttgcg ggggatccac agcctgacta actcgacaaa cccccccaaa cctaaggtga
     3301 gttgatcttt aagcttttta cattttcagc tcgcatatat caattcgcgc gcattgtcac
     3361 actagcaccc actagcggtc agactgcaga ttgcagcacg aaacaggaag ctgactccac
     3421 atggtcacat gctcactgaa gtgttgactt ccctgacagc tgtgcacttt ctaaaccggt
     3481 tttctcattc atttacagtt cagccggaat tcacctcgag accatggctc caaagaagaa
     3541 gcgtaaggta accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
     3601 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     3661 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     3721 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     3781 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     3841 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     3901 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     3961 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     4021 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac
     4081 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     4141 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     4201 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctca attaaagctg
     4261 agatctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4321 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4381 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4441 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     4501 gaaagaacca aagggcgaat taattcattg atgagtttgg acaaaccaca actagaatgc
     4561 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4621 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     4681 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattgatgag
     4741 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4801 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4861 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4921 ctctacaaat gtggtcattc agatcaatta accctcaccc gggctagcga tgcatgggag
     4981 ctcaagacgt ctgataactt cgtatagcat acattatacg aagttatcta cctaacttta
     5041 aataatgcca attatttaaa gttacttccg cgcccgggga gcccaagggc acgccctggc
     5101 acctgacgta cgttgtctag caaggaagtg aagagctatc cggatatata tgtatgttat
     5161 cataactgta ctggctatgt caggtggtaa tgactgtaac gttacgttac tcgttgtagg
     5221 cacgacattg aatatgctct tctccgctgt ggcccgaacg cctcccgcta gcgatgggat
     5281 ctaatacgac tcactatagg gagaccaagg gaaaatagaa tgaagtgatc tccaaaaaat
     5341 aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa
     5401 aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat
     5461 ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc
     5521 tgggatccag ctgcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     5581 ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat
     5641 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     5701 tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     5761 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     5821 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     5881 atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     5941 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     6001 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     6061 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     6121 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     6181 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     6241 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     6301 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     6361 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa
     6421 cactggcaaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6481 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6541 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6601 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6661 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6721 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6781 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6841 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6901 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacggtt cccgaaggga
     6961 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7021 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7081 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7141 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7201 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     7261 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaacag a
//
