LOCUS       sequence-380745-        7181 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 7181)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7181)
  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-cmlc2GFP.1
FEATURES             Location/Qualifiers
     source          1..7181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     224..241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(271..282,283..445)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    532..553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(568..573,574..591,592..598)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    606..622
                     /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     611..633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     630..646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             642..992
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSLLDNGPI
                     TLNNANYLKLLS"
     promoter        667..685
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(733..751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(761..778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(761..777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(770..792)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    952..983
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(989..1022)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1200..1256
                     /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             1263..1700
                     /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    4389..4613
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4683..4764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4888..4969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5118..5151)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5157..5188
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5193..5226
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5406..5424
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5447..5634,5635..5646)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5700..5719)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5711..6520
                     /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(5778..5797)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6663..7181,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7152..7171
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa ttaatgtgag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg
      721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
      781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      901 gcctgaatgg cgaatggacc tgccacactt ccttgctaga caatgggccc ataactttaa
      961 ataatgccaa ttatttaaag ttactatcat aacttcgtat agcatacatt atacgaagtt
     1021 atacctaggc ttaccaccca ctagcggtca gactgcagat tgcagcacga aacaggaagc
     1081 tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct gtgcactttc
     1141 taaaccggtt ttctcattca tttacagttc agccggaatt cacccgggta ccggaaggtg
     1201 ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc ggtccttcta
     1261 gaaagctact gtcttctatc gaacaagcat gcgatatttg ccgacttaaa aagctcaagt
     1321 gctccaaaga aaaaccgaag tgcgccaagt gtctgaagaa caactgggag tgtcgctact
     1381 ctcccaaaac caaaaggtct ccgctgacta gggcacatct gacagaagtg gaatcaaggc
     1441 tagaaagact ggaacagcta tttctactga tttttcctcg agaagacctt gacatgattt
     1501 tgaaaatgga ttctttacag gatataaaag cattgttaac aggattattt gtacaagata
     1561 atgtgaataa agatgccgtc acagatagat tggcttcagt ggagactgat atgcctctaa
     1621 cattgagaca gcatagaata agtgcgacat catcatcgga agagagtagt aacaaaggtc
     1681 aaagacagtt gactgtatcg tcgaggtcga ccccgggaat acagatctct cgagccgccc
     1741 ccccgaccga tgtcagcctg ggggacgagc tccacttaga cggcgaggac gtggcgatgg
     1801 cgcatgccga cgcgctagac gatttcgatc tggacatgtt gggggacggg gattccccgg
     1861 gtccgggatt taccccccac gactccgccc cctacggcgc tctggatatg taactagtaa
     1921 ttaagtctca gccaccgtta actgaacatg tcaaaacctg tggagactgt tgagatttga
     1981 tgttctgaaa agataaagcc tataaataaa atgttgccca aatttcctgc ctgatgtttt
     2041 tctttgtctt tgctacatgg ctttgctgct cggatcggct cactctgtgt atgccacgtt
     2101 cactttgtac tctccttctc acggtaggtt tattattttt agatgtgcag ttagtttctg
     2161 tgaaataaca caccacacac tgatattgtc tgtgcattga cttggtgagt gcacattgtt
     2221 tttgatcttg acatatttat atttgattga tcaggtgaac tgtgtgaatc taaagtgctc
     2281 catacagatg ttctgcattg aaaatattct cattttatta gtggaagtga gtgtatgcca
     2341 catccaatca atttcagcaa acaccccagt atgatttaat gcaaaaaaat gaaggtatca
     2401 aacacgcatt actactttgc agttaaatat ttaacattta ttccaacacg aaaaaaagca
     2461 gtaaataaca ctttgacaaa cacgtcagga catcttattt ttgtcaccct cacaggcaat
     2521 ttagtataat atattatata tatatatata tcatataata atattcagta taatatatat
     2581 atatatatca tattataata ttcagtataa tataaaacac aaacacatat atgtataata
     2641 taatataaca tttttattta ttgagatgcc tctatggacc gtgttataag aagtaaagat
     2701 caggagaagt aaacatgaag tgtaattatg aatactgatg ttaaattaag ctatgatgag
     2761 ttttcactgt taatttacca tctcaattaa atgttgatgc ctccatgacc aagttaagca
     2821 gatgagactg aggacaactg tagaagacaa gatgttcact ttgctgaata tagctggctt
     2881 gacagttatc tatgactcta taaatatata tatatttttt tttttataaa atgatttatt
     2941 tataactata tatccatttc tcagacaggt gcttcatatc cctcactccc gtagctgtcc
     3001 atgctggatc tgtccccgtt gtttttaaaa agctaaataa gttattaaca tgactgcatc
     3061 cagcgagcca aacctgtctg gtgtacagct accagagaag cttgagatcc tagtcaccgg
     3121 cggcattcat ccatcctttt catccctcaa atctctcatt cacgtccccc tccccatctg
     3181 cacactttat ctcattttcc accctgctgg aatctgagca cttgtgcagt tatcagggct
     3241 cctgtattta ggaggctctg ggtgtccatg taggggacga acagaaacac tgcagacctt
     3301 tatagaagaa caaatgataa gagtcctcat acataaagac tccattagaa acgtcagtga
     3361 cccaggagcc cagaccaaca gcaaagcaga cagtgaagga tccacagcct gactaactcg
     3421 acaaaccccc ccaaacctaa ggtgagttga tctttaagct ttttacattt tcagctcgca
     3481 tatatcaatt cgcgcgcatt gtcacactag cacccactag cggtcagact gcagattgca
     3541 gcacgaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt gacttccctg
     3601 acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc ggaattcctc
     3661 gagaccatga gcggaggtga ggagctgttc gccggcatcg tgcccgtgct gatcgagctg
     3721 gacggcgacg tgcacggcca caagttcagc gtgcgcggcg agggcgaggg cgacgccgac
     3781 tacggcaagc tggagatcaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3841 accctggtga ccaccctctg ctacggcatc cagtgcttcg cccgctaccc cgagcacatg
     3901 aagatgaacg acttcttcaa gagcgccatg cccgagggct acatccagga gcgcaccatc
     3961 cagttccagg acgacggcaa gtacaagacc cgcggcgagg tgaagttcga gggcgacacc
     4021 ctggtgaacc gcatcgagct gaagggcaag gacttcaagg aggacggcaa catcctgggc
     4081 cacaagctgg agtacagctt caacagccac aacgtgtaca tccgccccga caaggccaac
     4141 aacggcctgg aggctaactt caagacccgc cacaacatcg agggcggcgg cgtgcagctg
     4201 gccgaccact accagaccaa cgtgcccctg ggcgacggcc ccgtgctgat ccccatcaac
     4261 cactacctga gcactcagac caagatcagc aaggaccgca acgaggcccg cgaccacatg
     4321 gtgctcctgg agtccttcag cgcctgctgc cacacccacg gcatggacga gctgtatagg
     4381 taatagatct gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     4441 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4501 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4561 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4621 ggcggaaaga accaaagggc gaattaattc attgatgagt ttggacaaac cacaactaga
     4681 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4741 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4801 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattga
     4861 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4921 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     4981 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     5041 aaacctctac aaatgtggtc attcagatca attaaccctc acccgggcta gcgatgcatg
     5101 ggagctcaag acgtctgata acttcgtata gcatacatta tacgaagtta tctacctaac
     5161 tttaaataat gccaattatt taaagttact tccgcgcccg gggagcccaa gggcacgccc
     5221 tggcacctga cgtacgttgt ctagcaagga agtgaagagc tatccggata tatatgtatg
     5281 ttatcataac tgtactggct atgtcaggtg gtaatgactg taacgttacg ttactcgttg
     5341 taggcacgac attgaatatg ctcttctccg ctgtggcccg aacgcctccc gctagcgatg
     5401 ggatctaata cgactcacta tagggagacc aagggaaaat agaatgaagt gatctccaaa
     5461 aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta
     5521 aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa
     5581 aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca
     5641 cctctgggat ccagctgcat catgaacaat aaaactgtct gcttacataa acagtaatac
     5701 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     5761 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     5821 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     5881 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     5941 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     6001 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     6061 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     6121 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     6181 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     6241 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     6301 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     6361 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     6421 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     6481 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     6541 gtaacactgg caaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6601 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6661 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6721 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6781 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6841 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6901 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6961 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7021 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac ggttcccgaa
     7081 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7141 gagcttccag ggggaaacgc ctggtatctt tatagtcctg t
//
