LOCUS       sequence-381138-        7202 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 7202)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7202)
  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.1
FEATURES             Location/Qualifiers
     source          1..7202
                     /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"
     CDS             3675..3695
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3699..4400
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4410..4634
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4704..4785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4909..4990
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5139..5172)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5178..5209
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5214..5247
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5427..5445
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5468..5655,5656..5667)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5721..5740)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5732..6541
                     /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(5799..5818)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6684..7202,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7173..7192
                     /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 ggaattcacc
     3661 tcgagaccat ggctccaaag aagaagcgta aggtaaccat gagcgagctg attaaggaga
     3721 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     3781 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     3841 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     3901 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct
     3961 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     4021 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat
     4081 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt
     4141 accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga
     4201 gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca
     4261 agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg
     4321 agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac
     4381 tggggcacaa gctcaattaa agctgagatc tgtgccttct agttgccagc catctgttgt
     4441 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4501 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4561 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4621 ggtgggctct atggcttctg aggcggaaag aaccaaaggg cgaattaatt cattgatgag
     4681 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4741 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4801 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4861 ctctacaaat gtggtcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4921 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4981 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     5041 ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattcagatc aattaaccct
     5101 cacccgggct agcgatgcat gggagctcaa gacgtctgat aacttcgtat agcatacatt
     5161 atacgaagtt atctacctaa ctttaaataa tgccaattat ttaaagttac ttccgcgccc
     5221 ggggagccca agggcacgcc ctggcacctg acgtacgttg tctagcaagg aagtgaagag
     5281 ctatccggat atatatgtat gttatcataa ctgtactggc tatgtcaggt ggtaatgact
     5341 gtaacgttac gttactcgtt gtaggcacga cattgaatat gctcttctcc gctgtggccc
     5401 gaacgcctcc cgctagcgat gggatctaat acgactcact atagggagac caagggaaaa
     5461 tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     5521 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     5581 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     5641 aaattactca agtactttac acctctggga tccagctgca tcatgaacaa taaaactgtc
     5701 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     5761 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     5821 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     5881 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     5941 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     6001 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     6061 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     6121 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6181 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6241 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     6301 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     6361 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     6421 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     6481 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     6541 atcagaattg gttaattggt tgtaacactg gcaaaaagga tctaggtgaa gatccttttt
     6601 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6661 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6721 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6781 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6841 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6901 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6961 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7021 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7081 gaaagcgcca cggttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7141 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7201 gt
//
