LOCUS       sequence-381128-        7308 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 7308)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7308)
  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-insBFP.1
FEATURES             Location/Qualifiers
     source          1..7308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(17..28,29..191)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    278..299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(314..319,320..337,338..344)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    352..368
                     /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     357..379
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     376..392
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     376..392
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             388..738
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSLLDNGPI
                     TLNNANYLKLLS"
     promoter        413..431
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(479..497)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(507..524)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(507..523)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(516..538)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    698..729
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(735..768)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             946..1002
                     /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             1012..1449
                     /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             3527..3547
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3551..4252
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4262..4486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4556..4637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4761..4842
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4991..5024)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5030..5061
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5066..5099
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5279..5297
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5320..5507,5508..5519)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5573..5592)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5584..6393
                     /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(5651..5670)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6536..7124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7025..7044
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7278..7295
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttaaatggt accgagcaga ggtgtaaaaa gtactcaaaa attttactca agtgaaagta
       61 caagtactta gggaaaattt tactcaatta aaagtaaaag tatctggcta gaatcttact
      121 tgagtaaaag taaaaaagta ctccattaaa attgtacttg agtattaagg aagtaaaagt
      181 aaaagcaaga aagaaaacta gagtggtctc cctttagtga gggtcaattg atgggaggcg
      241 ttcgggccac agcggacacg caggtgcgca acgcaattaa tgtgagttag ctcactcatt
      301 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      361 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
      421 ctcactaaag ggaacaaaag ctggaggacc tcgagggggg gcccggtacc caattcgccc
      481 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
      541 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
      601 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
      661 tggacctgcc acacttcctt gctagacaat gggcccataa ctttaaataa tgccaattat
      721 ttaaagttac tatcataact tcgtatagca tacattatac gaagttatac ctaggcttac
      781 cacccactag cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt
      841 cacatgctca ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct
      901 cattcattta cagttcagcc ggaattcacc cgggtaccgg aaggtgccac gaacttctct
      961 ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctacgtacca gaagctactg
     1021 tcttctatcg aacaagcatg cgatatttgc cgacttaaaa agctcaagtg ctccaaagaa
     1081 aaaccgaagt gcgccaagtg tctgaagaac aactgggagt gtcgctactc tcccaaaacc
     1141 aaaaggtctc cgctgactag ggcacatctg acagaagtgg aatcaaggct agaaagactg
     1201 gaacagctat ttctactgat ttttcctcga gaagaccttg acatgatttt gaaaatggat
     1261 tctttacagg atataaaagc attgttaaca ggattatttg tacaagataa tgtgaataaa
     1321 gatgccgtca cagatagatt ggcttcagtg gagactgata tgcctctaac attgagacag
     1381 catagaataa gtgcgacatc atcatcggaa gagagtagta acaaaggtca aagacagttg
     1441 actgtatcgt cgaggtcgac cccgggaata cagatctctc gagccgcccc cccgaccgat
     1501 gtcagcctgg gggacgagct ccacttagac ggcgaggacg tggcgatggc gcatgccgac
     1561 gcgctagacg atttcgatct ggacatgttg ggggacgggg attccccggg tccgggattt
     1621 accccccacg actccgcccc ctacggcgct ctggatatgt aactagtaat taagtctcag
     1681 ccaccgttaa ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa
     1741 gataaagcct ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt
     1801 gctacatggc tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact
     1861 ctccttctca cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac
     1921 accacacact gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga
     1981 catatttata tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt
     2041 tctgcattga aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa
     2101 tttcagcaaa caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta
     2161 ctactttgca gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac
     2221 tttgacaaac acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata
     2281 tattatatat atatatatat catataataa tattcagtat aatatatata tatatatcat
     2341 attataatat tcagtataat ataaaacaca aacacatata tgtataatat aatataacat
     2401 ttttatttat tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta
     2461 aacatgaagt gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt
     2521 aatttaccat ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga
     2581 ggacaactgt agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct
     2641 atgactctat aaatatatat atattttttt ttttataaaa tgatttattt ataactatat
     2701 atccatttct cagacaggtg cttcatatcc ctcactcccg tagctgtcca tgctggatct
     2761 gtccccgttg tttttaaaaa gctaaataag ttattaacat gactgcatcc agcgagccaa
     2821 acctgtctgg tgtacagcta ccagagaagc catatgggcg cacaaaatga tctaatagta
     2881 tactataaaa tctttcaaaa tccaggccac taccctatga ctaaaaccac acatattagg
     2941 ttttaaaaaa aagtcacaat gaagacgtca caaacgttcc tgaagactaa tgactcgtct
     3001 aaccccatct taccttctgc tgtttcactc aaagaaaagc caccagctgt ctctgtgatc
     3061 tgctcattct gtgtaaactt gtgttctcaa caattaacaa ttaaccaaaa gcctctggag
     3121 ctgcgtgagc atcacgatgt gtttgtcttc tgtgagcgtg gcagtgtcca cgaagtcaat
     3181 gtgaattggt tggccaaaca cagctaatgt ggcatgtgtg attggacagc cacttaaaag
     3241 tgttaaagtg tgtttacgca caggattggt tttccgctga atcaggcaaa accagcagaa
     3301 ccgatctaaa ctcgagtata aaagtgagta ggagatgcta ttcgtcccaa aacatctcca
     3361 ccaccatatc caccattcct cgcctctgct tcgagaacag gtgagtgtcg agcgggatgg
     3421 taaatctaca gagaatgcgg agtgtagctt gtgtacatgt ttttgattaa cagagattgt
     3481 atgtgtgtgt ttgtgtcagt gtggaattca cctcgagacc atggctccaa agaagaagcg
     3541 taaggtaacc atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     3601 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     3661 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     3721 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     3781 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     3841 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     3901 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     3961 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     4021 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     4081 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     4141 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     4201 agtggccaga tactgcgacc tccctagcaa actggggcac aagctcaatt aaagctgaga
     4261 tctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4321 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     4381 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     4441 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     4501 agaaccaaag ggcgaattaa ttcattgatg agtttggaca aaccacaact agaatgcagt
     4561 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     4621 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     4681 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtcat tgatgagttt
     4741 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4801 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4861 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     4921 tacaaatgtg gtcattcaga tcaattaacc ctcacccggg ctagcgatgc atgggagctc
     4981 aagacgtctg ataacttcgt atagcataca ttatacgaag ttatctacct aactttaaat
     5041 aatgccaatt atttaaagtt acttccgcgc ccggggagcc caagggcacg ccctggcacc
     5101 tgacgtacgt tgtctagcaa ggaagtgaag agctatccgg atatatatgt atgttatcat
     5161 aactgtactg gctatgtcag gtggtaatga ctgtaacgtt acgttactcg ttgtaggcac
     5221 gacattgaat atgctcttct ccgctgtggc ccgaacgcct cccgctagcg atgggatcta
     5281 atacgactca ctatagggag accaagggaa aatagaatga agtgatctcc aaaaaataag
     5341 tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat
     5401 gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc
     5461 caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt acacctctgg
     5521 gatccagctg catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     5581 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct
     5641 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     5701 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     5761 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     5821 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     5881 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     5941 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     6001 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     6061 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     6121 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     6181 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     6241 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     6301 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     6361 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac
     6421 tggcaaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6481 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6541 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6601 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6661 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6721 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6781 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6841 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6901 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacggttccc gaagggagaa
     6961 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7021 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7081 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7141 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     7201 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     7261 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaacaga
//
