LOCUS       sequence-380751-        7246 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 7246)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7246)
  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.2
FEATURES             Location/Qualifiers
     source          1..7246
                     /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"
     polyA_signal    4454..4678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4748..4829
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4953..5034
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5183..5216)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5222..5253
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5258..5291
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5471..5489
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5512..5699,5700..5711)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5765..5784)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5776..6585
                     /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(5843..5862)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6728..7246,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7217..7236
                     /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 tcctcgagac catgagcgga ggtgaggagc tgttcgccgg catcgtgccc gtgctgatcg
     3781 agctggacgg cgacgtgcac ggccacaagt tcagcgtgcg cggcgagggc gagggcgacg
     3841 ccgactacgg caagctggag atcaagttca tctgcaccac cggcaagctg cccgtgccct
     3901 ggcccaccct ggtgaccacc ctctgctacg gcatccagtg cttcgcccgc taccccgagc
     3961 acatgaagat gaacgacttc ttcaagagcg ccatgcccga gggctacatc caggagcgca
     4021 ccatccagtt ccaggacgac ggcaagtaca agacccgcgg cgaggtgaag ttcgagggcg
     4081 acaccctggt gaaccgcatc gagctgaagg gcaaggactt caaggaggac ggcaacatcc
     4141 tgggccacaa gctggagtac agcttcaaca gccacaacgt gtacatccgc cccgacaagg
     4201 ccaacaacgg cctggaggct aacttcaaga cccgccacaa catcgagggc ggcggcgtgc
     4261 agctggccga ccactaccag accaacgtgc ccctgggcga cggccccgtg ctgatcccca
     4321 tcaaccacta cctgagcact cagaccaaga tcagcaagga ccgcaacgag gcccgcgacc
     4381 acatggtgct cctggagtcc ttcagcgcct gctgccacac ccacggcatg gacgagctgt
     4441 ataggtaata gatctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4501 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4561 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4621 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     4681 tctgaggcgg aaagaaccaa agggcgaatt aattcattga tgagtttgga caaaccacaa
     4741 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4801 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4861 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggtc
     4921 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     4981 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5041 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     5101 aagtaaaacc tctacaaatg tggtcattca gatcaattaa ccctcacccg ggctagcgat
     5161 gcatgggagc tcaagacgtc tgataacttc gtatagcata cattatacga agttatctac
     5221 ctaactttaa ataatgccaa ttatttaaag ttacttccgc gcccggggag cccaagggca
     5281 cgccctggca cctgacgtac gttgtctagc aaggaagtga agagctatcc ggatatatat
     5341 gtatgttatc ataactgtac tggctatgtc aggtggtaat gactgtaacg ttacgttact
     5401 cgttgtaggc acgacattga atatgctctt ctccgctgtg gcccgaacgc ctcccgctag
     5461 cgatgggatc taatacgact cactataggg agaccaaggg aaaatagaat gaagtgatct
     5521 ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt
     5581 ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt actcaagtaa
     5641 agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta ctcaagtact
     5701 ttacacctct gggatccagc tgcatcatga acaataaaac tgtctgctta cataaacagt
     5761 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc
     5821 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     5881 gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     5941 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     6001 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     6061 accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt
     6121 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     6181 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     6241 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     6301 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     6361 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     6421 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     6481 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     6541 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     6601 tggttgtaac actggcaaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6661 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6721 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6781 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6841 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6901 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6961 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7021 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7081 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacggttc
     7141 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7201 cgagggagct tccaggggga aacgcctggt atctttatag tcctgt
//
