LOCUS       sequence-381143-        7200 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 7200)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7200)
  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-cmlc2BFP.3
FEATURES             Location/Qualifiers
     source          1..7200
                     /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             1198..1254
                     /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             1261..1698
                     /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             3673..3693
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3697..4398
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4408..4632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4702..4783
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4907..4988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5137..5170)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5176..5207
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5212..5245
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5425..5443
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5466..5653,5654..5665)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5719..5738)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5730..6539
                     /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(5797..5816)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6682..7200,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7171..7190
                     /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 tttacagcag ccggaattca cccgggtacc ggaaggtgcc
     1201 acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg tccttctaga
     1261 aagctactgt cttctatcga acaagcatgc gatatttgcc gacttaaaaa gctcaagtgc
     1321 tccaaagaaa aaccgaagtg cgccaagtgt ctgaagaaca actgggagtg tcgctactct
     1381 cccaaaacca aaaggtctcc gctgactagg gcacatctga cagaagtgga atcaaggcta
     1441 gaaagactgg aacagctatt tctactgatt tttcctcgag aagaccttga catgattttg
     1501 aaaatggatt ctttacagga tataaaagca ttgttaacag gattatttgt acaagataat
     1561 gtgaataaag atgccgtcac agatagattg gcttcagtgg agactgatat gcctctaaca
     1621 ttgagacagc atagaataag tgcgacatca tcatcggaag agagtagtaa caaaggtcaa
     1681 agacagttga ctgtatcgtc gaggtcgacc ccgggaatac agatctctcg agccgccccc
     1741 ccgaccgatg tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg
     1801 catgccgacg cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt
     1861 ccgggattta ccccccacga ctccgccccc tacggcgctc tggatatgta actagtaatt
     1921 aagtctcagc caccgttaac tgaacatgtc aaaacctgtg gagactgttg agatttgatg
     1981 ttctgaaaag ataaagccta taaataaaat gttgcccaaa tttcctgcct gatgtttttc
     2041 tttgtctttg ctacatggct ttgctgctcg gatcggctca ctctgtgtat gccacgttca
     2101 ctttgtactc tccttctcac ggtaggttta ttatttttag atgtgcagtt agtttctgtg
     2161 aaataacaca ccacacactg atattgtctg tgcattgact tggtgagtgc acattgtttt
     2221 tgatcttgac atatttatat ttgattgatc aggtgaactg tgtgaatcta aagtgctcca
     2281 tacagatgtt ctgcattgaa aatattctca ttttattagt ggaagtgagt gtatgccaca
     2341 tccaatcaat ttcagcaaac accccagtat gatttaatgc aaaaaaatga aggtatcaaa
     2401 cacgcattac tactttgcag ttaaatattt aacatttatt ccaacacgaa aaaaagcagt
     2461 aaataacact ttgacaaaca cgtcaggaca tcttattttt gtcaccctca caggcaattt
     2521 agtataatat attatatata tatatatatc atataataat attcagtata atatatatat
     2581 atatatcata ttataatatt cagtataata taaaacacaa acacatatat gtataatata
     2641 atataacatt tttatttatt gagatgcctc tatggaccgt gttataagaa gtaaagatca
     2701 ggagaagtaa acatgaagtg taattatgaa tactgatgtt aaattaagct atgatgagtt
     2761 ttcactgtta atttaccatc tcaattaaat gttgatgcct ccatgaccaa gttaagcaga
     2821 tgagactgag gacaactgta gaagacaaga tgttcacttt gctgaatata gctggcttga
     2881 cagttatcta tgactctata aatatatata tatttttttt tttataaaat gatttattta
     2941 taactatata tccatttctc agacaggtgc ttcatatccc tcactcccgt agctgtccat
     3001 gctggatctg tccccgttgt ttttaaaaag ctaaataagt tattaacatg actgcatcca
     3061 gcgagccaaa cctgtctggt gtacagctac cagagaagct tgagatccta gtcaccggcg
     3121 gcattcatcc atccttttca tccctcaaat ctctcattca cgtccccctc cccatctgca
     3181 cactttatct cattttccac cctgctggaa tctgagcact tgtgcagtta tcagggctcc
     3241 tgtatttagg aggctctggg tgtccatgta ggggacgaac agaaacactg cagaccttta
     3301 tagaagaaca aatgataaga gtcctcatac ataaagactc cattagaaac gtcagtgacc
     3361 caggagccca gaccaacagc aaagcagaca gtgaaggatc cacagcctga ctaactcgac
     3421 aaaccccccc aaacctaagg tgagttgatc tttaagcttt ttacattttc agctcgcata
     3481 tatcaattcg cgcgcattgt cacactagca cccactagcg gtcagactgc agattgcagc
     3541 acgaaacagg aagctgactc cacatggtca catgctcact gaagtgttga cttccctgac
     3601 agctgtgcac tttctaaacc ggttttctca ttcatttaca gttcagccgg aattcacctc
     3661 gagaccatgg ctccaaagaa gaagcgtaag gtaaccatga gcgagctgat taaggagaac
     3721 atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc
     3781 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc
     3841 ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc
     3901 ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc
     3961 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
     4021 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc
     4081 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac
     4141 cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc
     4201 catctgatcg caaacatcaa gaccacatat agatccaaga aacccgctaa gaacctcaag
     4261 atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag
     4321 acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg
     4381 gggcacaagc tcaattaaag ctgagatctg tgccttctag ttgccagcca tctgttgttt
     4441 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4501 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4561 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     4621 tgggctctat ggcttctgag gcggaaagaa ccaaagggcg aattaattca ttgatgagtt
     4681 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     4741 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     4801 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     4861 ctacaaatgt ggtcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     4921 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4981 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     5041 aggtttttta aagcaagtaa aacctctaca aatgtggtca ttcagatcaa ttaaccctca
     5101 cccgggctag cgatgcatgg gagctcaaga cgtctgataa cttcgtatag catacattat
     5161 acgaagttat ctacctaact ttaaataatg ccaattattt aaagttactt ccgcgcccgg
     5221 ggagcccaag ggcacgccct ggcacctgac gtacgttgtc tagcaaggaa gtgaagagct
     5281 atccggatat atatgtatgt tatcataact gtactggcta tgtcaggtgg taatgactgt
     5341 aacgttacgt tactcgttgt aggcacgaca ttgaatatgc tcttctccgc tgtggcccga
     5401 acgcctcccg ctagcgatgg gatctaatac gactcactat agggagacca agggaaaata
     5461 gaatgaagtg atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta
     5521 aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa
     5581 ttgtactcaa gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa
     5641 attactcaag tactttacac ctctgggatc cagctgcatc atgaacaata aaactgtctg
     5701 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
     5761 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     5821 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     5881 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     5941 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     6001 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
     6061 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     6121 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     6181 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     6241 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     6301 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     6361 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     6421 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     6481 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     6541 cagaattggt taattggttg taacactggc aaaaaggatc taggtgaaga tcctttttga
     6601 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6661 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6721 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6781 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6841 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6901 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6961 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7021 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7081 aagcgccacg gttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7141 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
//
