LOCUS       sequence-381142-        7286 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 7286)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7286)
  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-insGFP.2
FEATURES             Location/Qualifiers
     source          1..7286
                     /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             945..1001
                     /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             1011..1448
                     /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"
     polyA_signal    4240..4464
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4534..4615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4739..4820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4969..5002)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5008..5039
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5044..5077
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5257..5275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5298..5485,5486..5497)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5551..5570)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5562..6371
                     /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(5629..5648)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6514..7102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7003..7022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7256..7273
                     /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 cagtcagccg gaattcaccc gggtaccgga aggtgccacg aacttctctc
      961 tgttaaagca agcaggagac gtggaagaaa accccggtcc tacgtaccag aagctactgt
     1021 cttctatcga acaagcatgc gatatttgcc gacttaaaaa gctcaagtgc tccaaagaaa
     1081 aaccgaagtg cgccaagtgt ctgaagaaca actgggagtg tcgctactct cccaaaacca
     1141 aaaggtctcc gctgactagg gcacatctga cagaagtgga atcaaggcta gaaagactgg
     1201 aacagctatt tctactgatt tttcctcgag aagaccttga catgattttg aaaatggatt
     1261 ctttacagga tataaaagca ttgttaacag gattatttgt acaagataat gtgaataaag
     1321 atgccgtcac agatagattg gcttcagtgg agactgatat gcctctaaca ttgagacagc
     1381 atagaataag tgcgacatca tcatcggaag agagtagtaa caaaggtcaa agacagttga
     1441 ctgtatcgtc gaggtcgacc ccgggaatac agatctctcg agccgccccc ccgaccgatg
     1501 tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg
     1561 cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta
     1621 ccccccacga ctccgccccc tacggcgctc tggatatgta actagtaatt aagtctcagc
     1681 caccgttaac tgaacatgtc aaaacctgtg gagactgttg agatttgatg ttctgaaaag
     1741 ataaagccta taaataaaat gttgcccaaa tttcctgcct gatgtttttc tttgtctttg
     1801 ctacatggct ttgctgctcg gatcggctca ctctgtgtat gccacgttca ctttgtactc
     1861 tccttctcac ggtaggttta ttatttttag atgtgcagtt agtttctgtg aaataacaca
     1921 ccacacactg atattgtctg tgcattgact tggtgagtgc acattgtttt tgatcttgac
     1981 atatttatat ttgattgatc aggtgaactg tgtgaatcta aagtgctcca tacagatgtt
     2041 ctgcattgaa aatattctca ttttattagt ggaagtgagt gtatgccaca tccaatcaat
     2101 ttcagcaaac accccagtat gatttaatgc aaaaaaatga aggtatcaaa cacgcattac
     2161 tactttgcag ttaaatattt aacatttatt ccaacacgaa aaaaagcagt aaataacact
     2221 ttgacaaaca cgtcaggaca tcttattttt gtcaccctca caggcaattt agtataatat
     2281 attatatata tatatatatc atataataat attcagtata atatatatat atatatcata
     2341 ttataatatt cagtataata taaaacacaa acacatatat gtataatata atataacatt
     2401 tttatttatt gagatgcctc tatggaccgt gttataagaa gtaaagatca ggagaagtaa
     2461 acatgaagtg taattatgaa tactgatgtt aaattaagct atgatgagtt ttcactgtta
     2521 atttaccatc tcaattaaat gttgatgcct ccatgaccaa gttaagcaga tgagactgag
     2581 gacaactgta gaagacaaga tgttcacttt gctgaatata gctggcttga cagttatcta
     2641 tgactctata aatatatata tatttttttt tttataaaat gatttattta taactatata
     2701 tccatttctc agacaggtgc ttcatatccc tcactcccgt agctgtccat gctggatctg
     2761 tccccgttgt ttttaaaaag ctaaataagt tattaacatg actgcatcca gcgagccaaa
     2821 cctgtctggt gtacagctac cagagaagcc atatgggcgc acaaaatgat ctaatagtat
     2881 actataaaat ctttcaaaat ccaggccact accctatgac taaaaccaca catattaggt
     2941 tttaaaaaaa agtcacaatg aagacgtcac aaacgttcct gaagactaat gactcgtcta
     3001 accccatctt accttctgct gtttcactca aagaaaagcc accagctgtc tctgtgatct
     3061 gctcattctg tgtaaacttg tgttctcaac aattaacaat taaccaaaag cctctggagc
     3121 tgcgtgagca tcacgatgtg tttgtcttct gtgagcgtgg cagtgtccac gaagtcaatg
     3181 tgaattggtt ggccaaacac agctaatgtg gcatgtgtga ttggacagcc acttaaaagt
     3241 gttaaagtgt gtttacgcac aggattggtt ttccgctgaa tcaggcaaaa ccagcagaac
     3301 cgatctaaac tcgagtataa aagtgagtag gagatgctat tcgtcccaaa acatctccac
     3361 caccatatcc accattcctc gcctctgctt cgagaacagg tgagtgtcga gcgggatggt
     3421 aaatctacag agaatgcgga gtgtagcttg tgtacatgtt tttgattaac agagattgta
     3481 tgtgtgtgtt tgtgtcagtg tggaattcct cgagaccatg agcggaggtg aggagctgtt
     3541 cgccggcatc gtgcccgtgc tgatcgagct ggacggcgac gtgcacggcc acaagttcag
     3601 cgtgcgcggc gagggcgagg gcgacgccga ctacggcaag ctggagatca agttcatctg
     3661 caccaccggc aagctgcccg tgccctggcc caccctggtg accaccctct gctacggcat
     3721 ccagtgcttc gcccgctacc ccgagcacat gaagatgaac gacttcttca agagcgccat
     3781 gcccgagggc tacatccagg agcgcaccat ccagttccag gacgacggca agtacaagac
     3841 ccgcggcgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcaa
     3901 ggacttcaag gaggacggca acatcctggg ccacaagctg gagtacagct tcaacagcca
     3961 caacgtgtac atccgccccg acaaggccaa caacggcctg gaggctaact tcaagacccg
     4021 ccacaacatc gagggcggcg gcgtgcagct ggccgaccac taccagacca acgtgcccct
     4081 gggcgacggc cccgtgctga tccccatcaa ccactacctg agcactcaga ccaagatcag
     4141 caaggaccgc aacgaggccc gcgaccacat ggtgctcctg gagtccttca gcgcctgctg
     4201 ccacacccac ggcatggacg agctgtatag gtaatagatc tgtgccttct agttgccagc
     4261 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4321 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4381 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     4441 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccaaaggg cgaattaatt
     4501 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     4561 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     4621 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     4681 caagtaaaac ctctacaaat gtggtcattg atgagtttgg acaaaccaca actagaatgc
     4741 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4801 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     4861 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattcagatc
     4921 aattaaccct cacccgggct agcgatgcat gggagctcaa gacgtctgat aacttcgtat
     4981 agcatacatt atacgaagtt atctacctaa ctttaaataa tgccaattat ttaaagttac
     5041 ttccgcgccc ggggagccca agggcacgcc ctggcacctg acgtacgttg tctagcaagg
     5101 aagtgaagag ctatccggat atatatgtat gttatcataa ctgtactggc tatgtcaggt
     5161 ggtaatgact gtaacgttac gttactcgtt gtaggcacga cattgaatat gctcttctcc
     5221 gctgtggccc gaacgcctcc cgctagcgat gggatctaat acgactcact atagggagac
     5281 caagggaaaa tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa
     5341 ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt
     5401 attgattttt aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag
     5461 taatcaagta aaattactca agtactttac acctctggga tccagctgca tcatgaacaa
     5521 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     5581 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     5641 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     5701 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     5761 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     5821 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     5881 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     5941 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     6001 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     6061 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     6121 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     6181 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     6241 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     6301 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     6361 agtttttcta atcagaattg gttaattggt tgtaacactg gcaaaaagga tctaggtgaa
     6421 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6481 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6541 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6601 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6661 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6721 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6781 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6841 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6901 tgagctatga gaaagcgcca cggttcccga agggagaaag gcggacaggt atccggtaag
     6961 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7021 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7081 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7141 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7201 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7261 gtcagtgagc gaggaagcgg aacaga
//
