LOCUS       sequence-381116-        7201 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 7201)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7201)
  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.2
FEATURES             Location/Qualifiers
     source          1..7201
                     /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             1199..1255
                     /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             1262..1699
                     /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             3674..3694
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3698..4399
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4409..4633
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4703..4784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4908..4989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5138..5171)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5177..5208
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5213..5246
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5426..5444
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5467..5654,5655..5666)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5720..5739)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5731..6540
                     /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(5798..5817)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6683..7201,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7172..7191
                     /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 tttacagtca gccggaattc acccgggtac cggaaggtgc
     1201 cacgaacttc tctctgttaa agcaagcagg agacgtggaa gaaaaccccg gtccttctag
     1261 aaagctactg tcttctatcg aacaagcatg cgatatttgc cgacttaaaa agctcaagtg
     1321 ctccaaagaa aaaccgaagt gcgccaagtg tctgaagaac aactgggagt gtcgctactc
     1381 tcccaaaacc aaaaggtctc cgctgactag ggcacatctg acagaagtgg aatcaaggct
     1441 agaaagactg gaacagctat ttctactgat ttttcctcga gaagaccttg acatgatttt
     1501 gaaaatggat tctttacagg atataaaagc attgttaaca ggattatttg tacaagataa
     1561 tgtgaataaa gatgccgtca cagatagatt ggcttcagtg gagactgata tgcctctaac
     1621 attgagacag catagaataa gtgcgacatc atcatcggaa gagagtagta acaaaggtca
     1681 aagacagttg actgtatcgt cgaggtcgac cccgggaata cagatctctc gagccgcccc
     1741 cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg tggcgatggc
     1801 gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg attccccggg
     1861 tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgt aactagtaat
     1921 taagtctcag ccaccgttaa ctgaacatgt caaaacctgt ggagactgtt gagatttgat
     1981 gttctgaaaa gataaagcct ataaataaaa tgttgcccaa atttcctgcc tgatgttttt
     2041 ctttgtcttt gctacatggc tttgctgctc ggatcggctc actctgtgta tgccacgttc
     2101 actttgtact ctccttctca cggtaggttt attattttta gatgtgcagt tagtttctgt
     2161 gaaataacac accacacact gatattgtct gtgcattgac ttggtgagtg cacattgttt
     2221 ttgatcttga catatttata tttgattgat caggtgaact gtgtgaatct aaagtgctcc
     2281 atacagatgt tctgcattga aaatattctc attttattag tggaagtgag tgtatgccac
     2341 atccaatcaa tttcagcaaa caccccagta tgatttaatg caaaaaaatg aaggtatcaa
     2401 acacgcatta ctactttgca gttaaatatt taacatttat tccaacacga aaaaaagcag
     2461 taaataacac tttgacaaac acgtcaggac atcttatttt tgtcaccctc acaggcaatt
     2521 tagtataata tattatatat atatatatat catataataa tattcagtat aatatatata
     2581 tatatatcat attataatat tcagtataat ataaaacaca aacacatata tgtataatat
     2641 aatataacat ttttatttat tgagatgcct ctatggaccg tgttataaga agtaaagatc
     2701 aggagaagta aacatgaagt gtaattatga atactgatgt taaattaagc tatgatgagt
     2761 tttcactgtt aatttaccat ctcaattaaa tgttgatgcc tccatgacca agttaagcag
     2821 atgagactga ggacaactgt agaagacaag atgttcactt tgctgaatat agctggcttg
     2881 acagttatct atgactctat aaatatatat atattttttt ttttataaaa tgatttattt
     2941 ataactatat atccatttct cagacaggtg cttcatatcc ctcactcccg tagctgtcca
     3001 tgctggatct gtccccgttg tttttaaaaa gctaaataag ttattaacat gactgcatcc
     3061 agcgagccaa acctgtctgg tgtacagcta ccagagaagc ttgagatcct agtcaccggc
     3121 ggcattcatc catccttttc atccctcaaa tctctcattc acgtccccct ccccatctgc
     3181 acactttatc tcattttcca ccctgctgga atctgagcac ttgtgcagtt atcagggctc
     3241 ctgtatttag gaggctctgg gtgtccatgt aggggacgaa cagaaacact gcagaccttt
     3301 atagaagaac aaatgataag agtcctcata cataaagact ccattagaaa cgtcagtgac
     3361 ccaggagccc agaccaacag caaagcagac agtgaaggat ccacagcctg actaactcga
     3421 caaacccccc caaacctaag gtgagttgat ctttaagctt tttacatttt cagctcgcat
     3481 atatcaattc gcgcgcattg tcacactagc acccactagc ggtcagactg cagattgcag
     3541 cacgaaacag gaagctgact ccacatggtc acatgctcac tgaagtgttg acttccctga
     3601 cagctgtgca ctttctaaac cggttttctc attcatttac agttcagccg gaattcacct
     3661 cgagaccatg gctccaaaga agaagcgtaa ggtaaccatg agcgagctga ttaaggagaa
     3721 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     3781 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     3841 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     3901 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     3961 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     4021 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     4081 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     4141 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag
     4201 ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa
     4261 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     4321 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     4381 ggggcacaag ctcaattaaa gctgagatct gtgccttcta gttgccagcc atctgttgtt
     4441 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     4501 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     4561 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     4621 gtgggctcta tggcttctga ggcggaaaga accaaagggc gaattaattc attgatgagt
     4681 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     4741 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     4801 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     4861 tctacaaatg tggtcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4921 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4981 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     5041 gaggtttttt aaagcaagta aaacctctac aaatgtggtc attcagatca attaaccctc
     5101 acccgggcta gcgatgcatg ggagctcaag acgtctgata acttcgtata gcatacatta
     5161 tacgaagtta tctacctaac tttaaataat gccaattatt taaagttact tccgcgcccg
     5221 gggagcccaa gggcacgccc tggcacctga cgtacgttgt ctagcaagga agtgaagagc
     5281 tatccggata tatatgtatg ttatcataac tgtactggct atgtcaggtg gtaatgactg
     5341 taacgttacg ttactcgttg taggcacgac attgaatatg ctcttctccg ctgtggcccg
     5401 aacgcctccc gctagcgatg ggatctaata cgactcacta tagggagacc aagggaaaat
     5461 agaatgaagt gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt
     5521 aaaaagtact tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta
     5581 attgtactca agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa
     5641 aattactcaa gtactttaca cctctgggat ccagctgcat catgaacaat aaaactgtct
     5701 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg
     5761 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     5821 gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg
     5881 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     5941 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     6001 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa
     6061 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     6121 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     6181 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     6241 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     6301 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     6361 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     6421 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     6481 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     6541 tcagaattgg ttaattggtt gtaacactgg caaaaaggat ctaggtgaag atcctttttg
     6601 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6661 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6721 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6781 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6841 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6901 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6961 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7021 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7081 aaagcgccac ggttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7141 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7201 t
//
