LOCUS       sequence-381118-        7165 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 7165)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7165)
  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-cmlc2BFP.1
FEATURES             Location/Qualifiers
     source          1..7165
                     /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             3638..3658
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3662..4363
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4373..4597
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4667..4748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4872..4953
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5102..5135)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5141..5172
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5177..5210
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5390..5408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5431..5618,5619..5630)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5684..5703)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5695..6504
                     /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(5762..5781)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6647..7165,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7136..7155
                     /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 aagcttgaga tcctagtcac cggcggcatt catccatcct tttcatccct caaatctctc
     3121 attcacgtcc ccctccccat ctgcacactt tatctcattt tccaccctgc tggaatctga
     3181 gcacttgtgc agttatcagg gctcctgtat ttaggaggct ctgggtgtcc atgtagggga
     3241 cgaacagaaa cactgcagac ctttatagaa gaacaaatga taagagtcct catacataaa
     3301 gactccatta gaaacgtcag tgacccagga gcccagacca acagcaaagc agacagtgaa
     3361 ggatccacag cctgactaac tcgacaaacc cccccaaacc taaggtgagt tgatctttaa
     3421 gctttttaca ttttcagctc gcatatatca attcgcgcgc attgtcacac tagcacccac
     3481 tagcggtcag actgcagatt gcagcacgaa acaggaagct gactccacat ggtcacatgc
     3541 tcactgaagt gttgacttcc ctgacagctg tgcactttct aaaccggttt tctcattcat
     3601 ttacagttca gccggaattc acctcgagac catggctcca aagaagaagc gtaaggtaac
     3661 catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     3721 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     3781 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     3841 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     3901 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
     3961 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     4021 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     4081 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     4141 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
     4201 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     4261 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
     4321 atactgcgac ctccctagca aactggggca caagctcaat taaagctgag atctgtgcct
     4381 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     4441 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     4501 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     4561 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccaaa
     4621 gggcgaatta attcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     4681 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4741 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     4801 aggtttttta aagcaagtaa aacctctaca aatgtggtca ttgatgagtt tggacaaacc
     4861 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4921 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4981 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     5041 ggtcattcag atcaattaac cctcacccgg gctagcgatg catgggagct caagacgtct
     5101 gataacttcg tatagcatac attatacgaa gttatctacc taactttaaa taatgccaat
     5161 tatttaaagt tacttccgcg cccggggagc ccaagggcac gccctggcac ctgacgtacg
     5221 ttgtctagca aggaagtgaa gagctatccg gatatatatg tatgttatca taactgtact
     5281 ggctatgtca ggtggtaatg actgtaacgt tacgttactc gttgtaggca cgacattgaa
     5341 tatgctcttc tccgctgtgg cccgaacgcc tcccgctagc gatgggatct aatacgactc
     5401 actataggga gaccaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg
     5461 actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag
     5521 taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa
     5581 tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg ggatccagct
     5641 gcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     5701 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     5761 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     5821 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     5881 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     5941 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     6001 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     6061 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     6121 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     6181 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     6241 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     6301 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     6361 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     6421 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     6481 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcaaaaa
     6541 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6601 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6661 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6721 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6781 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6841 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6901 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6961 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7021 gatacctaca gcgtgagcta tgagaaagcg ccacggttcc cgaagggaga aaggcggaca
     7081 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7141 acgcctggta tctttatagt cctgt
//
