LOCUS       sequence-380746-        7268 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 7268)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7268)
  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.1
FEATURES             Location/Qualifiers
     source          1..7268
                     /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..1874
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1200..1874
                     /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(1327..1345)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1595..1614
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1789..1812
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3741..3761
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3765..4466
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4476..4700
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4770..4851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4975..5056
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5205..5238)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5244..5275
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5280..5313
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5493..5511
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5534..5721,5722..5733)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5787..5806)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5798..6607
                     /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(5865..5884)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6750..7268,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7239..7258
                     /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 cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg
     1261 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     1321 agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
     1381 cccctcagtt ccagtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     1441 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     1501 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     1561 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     1621 gctgggaggc ctccaccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     1681 agatgaggct gaagctgaag gacggcggcc actacgacgc cgaggtcaag accacctaca
     1741 tggccaagaa gcccgtgcag ctgcccggcg cctacaagac cgacatcaag ctggacatca
     1801 cctcccacaa cgaggactac accatcgtgg aacagtacga gcgcgccgag ggccgccact
     1861 ccaccggagc ctaagactag taattaagtc tcagccaccg ttaactgaac atgtcaaaac
     1921 ctgtggagac tgttgagatt tgatgttctg aaaagataaa gcctataaat aaaatgttgc
     1981 ccaaatttcc tgcctgatgt ttttctttgt ctttgctaca tggctttgct gctcggatcg
     2041 gctcactctg tgtatgccac gttcactttg tactctcctt ctcacggtag gtttattatt
     2101 tttagatgtg cagttagttt ctgtgaaata acacaccaca cactgatatt gtctgtgcat
     2161 tgacttggtg agtgcacatt gtttttgatc ttgacatatt tatatttgat tgatcaggtg
     2221 aactgtgtga atctaaagtg ctccatacag atgttctgca ttgaaaatat tctcatttta
     2281 ttagtggaag tgagtgtatg ccacatccaa tcaatttcag caaacacccc agtatgattt
     2341 aatgcaaaaa aatgaaggta tcaaacacgc attactactt tgcagttaaa tatttaacat
     2401 ttattccaac acgaaaaaaa gcagtaaata acactttgac aaacacgtca ggacatctta
     2461 tttttgtcac cctcacaggc aatttagtat aatatattat atatatatat atatcatata
     2521 ataatattca gtataatata tatatatata tcatattata atattcagta taatataaaa
     2581 cacaaacaca tatatgtata atataatata acatttttat ttattgagat gcctctatgg
     2641 accgtgttat aagaagtaaa gatcaggaga agtaaacatg aagtgtaatt atgaatactg
     2701 atgttaaatt aagctatgat gagttttcac tgttaattta ccatctcaat taaatgttga
     2761 tgcctccatg accaagttaa gcagatgaga ctgaggacaa ctgtagaaga caagatgttc
     2821 actttgctga atatagctgg cttgacagtt atctatgact ctataaatat atatatattt
     2881 ttttttttat aaaatgattt atttataact atatatccat ttctcagaca ggtgcttcat
     2941 atccctcact cccgtagctg tccatgctgg atctgtcccc gttgttttta aaaagctaaa
     3001 taagttatta acatgactgc atccagcgag ccaaacctgt ctggtgtaca gctaccagag
     3061 aagccatatg ggcgcacaaa atgatctaat agtatactat aaaatctttc aaaatccagg
     3121 ccactaccct atgactaaaa ccacacatat taggttttaa aaaaaagtca caatgaagac
     3181 gtcacaaacg ttcctgaaga ctaatgactc gtctaacccc atcttacctt ctgctgtttc
     3241 actcaaagaa aagccaccag ctgtctctgt gatctgctca ttctgtgtaa acttgtgttc
     3301 tcaacaatta acaattaacc aaaagcctct ggagctgcgt gagcatcacg atgtgtttgt
     3361 cttctgtgag cgtggcagtg tccacgaagt caatgtgaat tggttggcca aacacagcta
     3421 atgtggcatg tgtgattgga cagccactta aaagtgttaa agtgtgttta cgcacaggat
     3481 tggttttccg ctgaatcagg caaaaccagc agaaccgatc taaactcgag tataaaagtg
     3541 agtaggagat gctattcgtc ccaaaacatc tccaccacca tatccaccat tcctcgcctc
     3601 tgcttcgaga acaggtgagt gtcgagcggg atggtaaatc tacagagaat gcggagtgta
     3661 gcttgtgtac atgtttttga ttaacagaga ttgtatgtgt gtgtttgtgt cagtgtggaa
     3721 ttcacctcga gaccatggct ccaaagaaga agcgtaaggt aaccatgagc gagctgatta
     3781 aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt
     3841 gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg
     3901 tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca
     3961 gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg
     4021 agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc
     4081 aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact
     4141 tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga
     4201 cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg
     4261 gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa cccgctaaga
     4321 acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca
     4381 acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta
     4441 gcaaactggg gcacaagctc aattaaagct gagatctgtg ccttctagtt gccagccatc
     4501 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     4561 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     4621 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     4681 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc aaagggcgaa ttaattcatt
     4741 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     4801 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4861 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4921 taaaacctct acaaatgtgg tcattgatga gtttggacaa accacaacta gaatgcagtg
     4981 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     5041 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     5101 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtcatt cagatcaatt
     5161 aaccctcacc cgggctagcg atgcatggga gctcaagacg tctgataact tcgtatagca
     5221 tacattatac gaagttatct acctaacttt aaataatgcc aattatttaa agttacttcc
     5281 gcgcccgggg agcccaaggg cacgccctgg cacctgacgt acgttgtcta gcaaggaagt
     5341 gaagagctat ccggatatat atgtatgtta tcataactgt actggctatg tcaggtggta
     5401 atgactgtaa cgttacgtta ctcgttgtag gcacgacatt gaatatgctc ttctccgctg
     5461 tggcccgaac gcctcccgct agcgatggga tctaatacga ctcactatag ggagaccaag
     5521 ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt
     5581 aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg
     5641 atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat
     5701 caagtaaaat tactcaagta ctttacacct ctgggatcca gctgcatcat gaacaataaa
     5761 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     5821 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     5881 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     5941 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     6001 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     6061 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     6121 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     6181 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     6241 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     6301 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     6361 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     6421 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     6481 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     6541 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     6601 tttctaatca gaattggtta attggttgta acactggcaa aaaggatcta ggtgaagatc
     6661 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6721 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6781 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6841 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6901 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6961 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7021 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7081 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7141 ctatgagaaa gcgccacggt tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7201 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7261 agtcctgt
//
