LOCUS       sequence-380895-        7179 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 7179)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7179)
  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.3
FEATURES             Location/Qualifiers
     source          1..7179
                     /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"
     polyA_signal    4387..4611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4681..4762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4886..4967
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5116..5149)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5155..5186
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5191..5224
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5404..5422
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5445..5632,5633..5644)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5698..5717)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5709..6518
                     /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(5776..5795)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6661..7179,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7150..7169
                     /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 aattcctcga
     3661 gaccatgagc ggaggtgagg agctgttcgc cggcatcgtg cccgtgctga tcgagctgga
     3721 cggcgacgtg cacggccaca agttcagcgt gcgcggcgag ggcgagggcg acgccgacta
     3781 cggcaagctg gagatcaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     3841 cctggtgacc accctctgct acggcatcca gtgcttcgcc cgctaccccg agcacatgaa
     3901 gatgaacgac ttcttcaaga gcgccatgcc cgagggctac atccaggagc gcaccatcca
     3961 gttccaggac gacggcaagt acaagacccg cggcgaggtg aagttcgagg gcgacaccct
     4021 ggtgaaccgc atcgagctga agggcaagga cttcaaggag gacggcaaca tcctgggcca
     4081 caagctggag tacagcttca acagccacaa cgtgtacatc cgccccgaca aggccaacaa
     4141 cggcctggag gctaacttca agacccgcca caacatcgag ggcggcggcg tgcagctggc
     4201 cgaccactac cagaccaacg tgcccctggg cgacggcccc gtgctgatcc ccatcaacca
     4261 ctacctgagc actcagacca agatcagcaa ggaccgcaac gaggcccgcg accacatggt
     4321 gctcctggag tccttcagcg cctgctgcca cacccacggc atggacgagc tgtataggta
     4381 atagatctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4441 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4501 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4561 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     4621 cggaaagaac caaagggcga attaattcat tgatgagttt ggacaaacca caactagaat
     4681 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4741 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4801 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattgatg
     4861 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4921 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4981 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     5041 acctctacaa atgtggtcat tcagatcaat taaccctcac ccgggctagc gatgcatggg
     5101 agctcaagac gtctgataac ttcgtatagc atacattata cgaagttatc tacctaactt
     5161 taaataatgc caattattta aagttacttc cgcgcccggg gagcccaagg gcacgccctg
     5221 gcacctgacg tacgttgtct agcaaggaag tgaagagcta tccggatata tatgtatgtt
     5281 atcataactg tactggctat gtcaggtggt aatgactgta acgttacgtt actcgttgta
     5341 ggcacgacat tgaatatgct cttctccgct gtggcccgaa cgcctcccgc tagcgatggg
     5401 atctaatacg actcactata gggagaccaa gggaaaatag aatgaagtga tctccaaaaa
     5461 ataagtactt tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa
     5521 aaaatgtaat taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa
     5581 atccccaaaa ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc
     5641 tctgggatcc agctgcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     5701 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     5761 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     5821 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     5881 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     5941 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     6001 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     6061 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     6121 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     6181 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     6241 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     6301 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     6361 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     6421 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     6481 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     6541 aacactggca aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6601 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6661 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6721 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6781 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6841 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6901 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6961 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     7021 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgg ttcccgaagg
     7081 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7141 gcttccaggg ggaaacgcct ggtatcttta tagtcctgt
//
