LOCUS       sequence-381122-        7247 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 7247)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7247)
  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-insGFP.1
FEATURES             Location/Qualifiers
     source          1..7247
                     /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"
     polyA_signal    4455..4679
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4749..4830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4954..5035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5184..5217)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5223..5254
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5259..5292
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5472..5490
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5513..5700,5701..5712)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5766..5785)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5777..6586
                     /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(5844..5863)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6729..7247,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7218..7237
                     /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 ttcctcgaga ccatgagcgg aggtgaggag ctgttcgccg gcatcgtgcc cgtgctgatc
     3781 gagctggacg gcgacgtgca cggccacaag ttcagcgtgc gcggcgaggg cgagggcgac
     3841 gccgactacg gcaagctgga gatcaagttc atctgcacca ccggcaagct gcccgtgccc
     3901 tggcccaccc tggtgaccac cctctgctac ggcatccagt gcttcgcccg ctaccccgag
     3961 cacatgaaga tgaacgactt cttcaagagc gccatgcccg agggctacat ccaggagcgc
     4021 accatccagt tccaggacga cggcaagtac aagacccgcg gcgaggtgaa gttcgagggc
     4081 gacaccctgg tgaaccgcat cgagctgaag ggcaaggact tcaaggagga cggcaacatc
     4141 ctgggccaca agctggagta cagcttcaac agccacaacg tgtacatccg ccccgacaag
     4201 gccaacaacg gcctggaggc taacttcaag acccgccaca acatcgaggg cggcggcgtg
     4261 cagctggccg accactacca gaccaacgtg cccctgggcg acggccccgt gctgatcccc
     4321 atcaaccact acctgagcac tcagaccaag atcagcaagg accgcaacga ggcccgcgac
     4381 cacatggtgc tcctggagtc cttcagcgcc tgctgccaca cccacggcat ggacgagctg
     4441 tataggtaat agatctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4501 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4561 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4621 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     4681 ttctgaggcg gaaagaacca aagggcgaat taattcattg atgagtttgg acaaaccaca
     4741 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     4801 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     4861 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     4921 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     4981 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     5041 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     5101 caagtaaaac ctctacaaat gtggtcattc agatcaatta accctcaccc gggctagcga
     5161 tgcatgggag ctcaagacgt ctgataactt cgtatagcat acattatacg aagttatcta
     5221 cctaacttta aataatgcca attatttaaa gttacttccg cgcccgggga gcccaagggc
     5281 acgccctggc acctgacgta cgttgtctag caaggaagtg aagagctatc cggatatata
     5341 tgtatgttat cataactgta ctggctatgt caggtggtaa tgactgtaac gttacgttac
     5401 tcgttgtagg cacgacattg aatatgctct tctccgctgt ggcccgaacg cctcccgcta
     5461 gcgatgggat ctaatacgac tcactatagg gagaccaagg gaaaatagaa tgaagtgatc
     5521 tccaaaaaat aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt
     5581 tttctaaaaa aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta
     5641 aagtaaaaat ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac
     5701 tttacacctc tgggatccag ctgcatcatg aacaataaaa ctgtctgctt acataaacag
     5761 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     5821 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     5881 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     5941 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     6001 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     6061 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     6121 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     6181 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     6241 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     6301 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     6361 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     6421 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     6481 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     6541 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     6601 ttggttgtaa cactggcaaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6661 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6721 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6781 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6841 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6901 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6961 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7021 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7081 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacggtt
     7141 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7201 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgt
//
