LOCUS       sequence-381105-        7264 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 7264)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7264)
  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-R-insBFP.2
FEATURES             Location/Qualifiers
     source          1..7264
                     /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..1873
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1199..1873
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(1326..1344)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1594..1613
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1788..1811
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3740..3760
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3764..4465
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4475..4699
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4769..4850
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4974..5055
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5201..5234)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5240..5271
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5276..5309
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5489..5507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5530..5717,5718..5729)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5783..5802)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5794..6603
                     /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(5861..5880)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6746..7264,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7235..7254
                     /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 ctcctccgag gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt
     1261 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     1321 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc
     1381 ccctcagttc cagtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     1441 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     1501 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     1561 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     1621 ctgggaggcc tccaccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     1681 gatgaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga ccacctacat
     1741 ggccaagaag cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc tggacatcac
     1801 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg gccgccactc
     1861 caccggagcc taagactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc
     1921 tgtggagact gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc
     1981 caaatttcct gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg
     2041 ctcactctgt gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt
     2101 ttagatgtgc agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt
     2161 gacttggtga gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga
     2221 actgtgtgaa tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat
     2281 tagtggaagt gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta
     2341 atgcaaaaaa atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt
     2401 tattccaaca cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat
     2461 ttttgtcacc ctcacaggca atttagtata atatattata tatatatata tatcatataa
     2521 taatattcag tataatatat atatatatat catattataa tattcagtat aatataaaac
     2581 acaaacacat atatgtataa tataatataa catttttatt tattgagatg cctctatgga
     2641 ccgtgttata agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga
     2701 tgttaaatta agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat
     2761 gcctccatga ccaagttaag cagatgagac tgaggacaac tgtagaagac aagatgttca
     2821 ctttgctgaa tatagctggc ttgacagtta tctatgactc tataaatata tatatatttt
     2881 tttttttata aaatgattta tttataacta tatatccatt tctcagacag gtgcttcata
     2941 tccctcactc ccgtagctgt ccatgctgga tctgtccccg ttgtttttaa aaagctaaat
     3001 aagttattaa catgactgca tccagcgagc caaacctgtc tggtgtacag ctaccagaga
     3061 agccatatgg gcgcacaaaa tgatctaata gtatactata aaatctttca aaatccaggc
     3121 cactacccta tgactaaaac cacacatatt aggttttaaa aaaaagtcac aatgaagacg
     3181 tcacaaacgt tcctgaagac taatgactcg tctaacccca tcttaccttc tgctgtttca
     3241 ctcaaagaaa agccaccagc tgtctctgtg atctgctcat tctgtgtaaa cttgtgttct
     3301 caacaattaa caattaacca aaagcctctg gagctgcgtg agcatcacga tgtgtttgtc
     3361 ttctgtgagc gtggcagtgt ccacgaagtc aatgtgaatt ggttggccaa acacagctaa
     3421 tgtggcatgt gtgattggac agccacttaa aagtgttaaa gtgtgtttac gcacaggatt
     3481 ggttttccgc tgaatcaggc aaaaccagca gaaccgatct aaactcgagt ataaaagtga
     3541 gtaggagatg ctattcgtcc caaaacatct ccaccaccat atccaccatt cctcgcctct
     3601 gcttcgagaa caggtgagtg tcgagcggga tggtaaatct acagagaatg cggagtgtag
     3661 cttgtgtaca tgtttttgat taacagagat tgtatgtgtg tgtttgtgtc agtgtggaat
     3721 tcacctcgag accatggctc caaagaagaa gcgtaaggta accatgagcg agctgattaa
     3781 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
     3841 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
     3901 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
     3961 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
     4021 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     4081 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     4141 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac
     4201 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
     4261 cgggagccat ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa
     4321 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
     4381 caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
     4441 caaactgggg cacaagctca attaaagctg agatctgtgc cttctagttg ccagccatct
     4501 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4561 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4621 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4681 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca aagggcgaat taattcattg
     4741 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     4801 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     4861 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     4921 aaaacctcta caaatgtggt cattgatgag tttggacaaa ccacaactag aatgcagtga
     4981 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     5041 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     5101 gtggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattcaga tcaattaacc
     5161 ctcacccggg ctagcgatgc atgggagctc aagacgtctg ataacttcgt atagcataca
     5221 ttatacgaag ttatctacct aactttaaat aatgccaatt atttaaagtt acttccgcgc
     5281 ccggggagcc caagggcacg ccctggcacc tgacgtacgt tgtctagcaa ggaagtgaag
     5341 agctatccgg atatatatgt atgttatcat aactgtactg gctatgtcag gtggtaatga
     5401 ctgtaacgtt acgttactcg ttgtaggcac gacattgaat atgctcttct ccgctgtggc
     5461 ccgaacgcct cccgctagcg atgggatcta atacgactca ctatagggag accaagggaa
     5521 aatagaatga agtgatctcc aaaaaataag tactttttga ctgtaaataa aattgtaagg
     5581 agtaaaaagt actttttttt ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt
     5641 ttaattgtac tcaagtaaag taaaaatccc caaaaataat acttaagtac agtaatcaag
     5701 taaaattact caagtacttt acacctctgg gatccagctg catcatgaac aataaaactg
     5761 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     5821 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     5881 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     5941 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     6001 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     6061 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     6121 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     6181 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     6241 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     6301 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     6361 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     6421 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     6481 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     6541 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     6601 taatcagaat tggttaattg gttgtaacac tggcaaaaag gatctaggtg aagatccttt
     6661 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6721 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6781 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6841 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6901 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6961 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7021 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7081 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7141 gagaaagcgc cacggttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7201 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7261 ctgt
//
