LOCUS       sequence-380743-        7245 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 7245)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7245)
  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.3
FEATURES             Location/Qualifiers
     source          1..7245
                     /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             1198..1872
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1198..1872
                     /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(1325..1343)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1593..1612
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1787..1810
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4453..4677
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4747..4828
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4952..5033
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5182..5215)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5221..5252
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5257..5290
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5470..5488
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5511..5698,5699..5710)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5764..5783)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5775..6584
                     /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(5842..5861)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6727..7245,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7216..7235
                     /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 tttacagcag ccggaattca cccgggtacc ggaaggtgcc
     1201 tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg
     1261 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1321 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1381 cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1441 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1501 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1561 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1621 tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1681 atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacatg
     1741 gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct ggacatcacc
     1801 tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc
     1861 accggagcct aagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct
     1921 gtggagactg ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc
     1981 aaatttcctg cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc
     2041 tcactctgtg tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt
     2101 tagatgtgca gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg
     2161 acttggtgag tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa
     2221 ctgtgtgaat ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt
     2281 agtggaagtg agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa
     2341 tgcaaaaaaa tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt
     2401 attccaacac gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt
     2461 tttgtcaccc tcacaggcaa tttagtataa tatattatat atatatatat atcatataat
     2521 aatattcagt ataatatata tatatatatc atattataat attcagtata atataaaaca
     2581 caaacacata tatgtataat ataatataac atttttattt attgagatgc ctctatggac
     2641 cgtgttataa gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat
     2701 gttaaattaa gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg
     2761 cctccatgac caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac
     2821 tttgctgaat atagctggct tgacagttat ctatgactct ataaatatat atatattttt
     2881 ttttttataa aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat
     2941 ccctcactcc cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata
     3001 agttattaac atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa
     3061 gccatatggg cgcacaaaat gatctaatag tatactataa aatctttcaa aatccaggcc
     3121 actaccctat gactaaaacc acacatatta ggttttaaaa aaaagtcaca atgaagacgt
     3181 cacaaacgtt cctgaagact aatgactcgt ctaaccccat cttaccttct gctgtttcac
     3241 tcaaagaaaa gccaccagct gtctctgtga tctgctcatt ctgtgtaaac ttgtgttctc
     3301 aacaattaac aattaaccaa aagcctctgg agctgcgtga gcatcacgat gtgtttgtct
     3361 tctgtgagcg tggcagtgtc cacgaagtca atgtgaattg gttggccaaa cacagctaat
     3421 gtggcatgtg tgattggaca gccacttaaa agtgttaaag tgtgtttacg cacaggattg
     3481 gttttccgct gaatcaggca aaaccagcag aaccgatcta aactcgagta taaaagtgag
     3541 taggagatgc tattcgtccc aaaacatctc caccaccata tccaccattc ctcgcctctg
     3601 cttcgagaac aggtgagtgt cgagcgggat ggtaaatcta cagagaatgc ggagtgtagc
     3661 ttgtgtacat gtttttgatt aacagagatt gtatgtgtgt gtttgtgtca gtgtggaatt
     3721 cctcgagacc atgagcggag gtgaggagct gttcgccggc atcgtgcccg tgctgatcga
     3781 gctggacggc gacgtgcacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgacgc
     3841 cgactacggc aagctggaga tcaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3901 gcccaccctg gtgaccaccc tctgctacgg catccagtgc ttcgcccgct accccgagca
     3961 catgaagatg aacgacttct tcaagagcgc catgcccgag ggctacatcc aggagcgcac
     4021 catccagttc caggacgacg gcaagtacaa gacccgcggc gaggtgaagt tcgagggcga
     4081 caccctggtg aaccgcatcg agctgaaggg caaggacttc aaggaggacg gcaacatcct
     4141 gggccacaag ctggagtaca gcttcaacag ccacaacgtg tacatccgcc ccgacaaggc
     4201 caacaacggc ctggaggcta acttcaagac ccgccacaac atcgagggcg gcggcgtgca
     4261 gctggccgac cactaccaga ccaacgtgcc cctgggcgac ggccccgtgc tgatccccat
     4321 caaccactac ctgagcactc agaccaagat cagcaaggac cgcaacgagg cccgcgacca
     4381 catggtgctc ctggagtcct tcagcgcctg ctgccacacc cacggcatgg acgagctgta
     4441 taggtaatag atctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     4501 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     4561 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     4621 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     4681 ctgaggcgga aagaaccaaa gggcgaatta attcattgat gagtttggac aaaccacaac
     4741 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4801 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4861 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtca
     4921 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4981 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     5041 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     5101 agtaaaacct ctacaaatgt ggtcattcag atcaattaac cctcacccgg gctagcgatg
     5161 catgggagct caagacgtct gataacttcg tatagcatac attatacgaa gttatctacc
     5221 taactttaaa taatgccaat tatttaaagt tacttccgcg cccggggagc ccaagggcac
     5281 gccctggcac ctgacgtacg ttgtctagca aggaagtgaa gagctatccg gatatatatg
     5341 tatgttatca taactgtact ggctatgtca ggtggtaatg actgtaacgt tacgttactc
     5401 gttgtaggca cgacattgaa tatgctcttc tccgctgtgg cccgaacgcc tcccgctagc
     5461 gatgggatct aatacgactc actataggga gaccaaggga aaatagaatg aagtgatctc
     5521 caaaaaataa gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt
     5581 tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa
     5641 gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt
     5701 tacacctctg ggatccagct gcatcatgaa caataaaact gtctgcttac ataaacagta
     5761 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     5821 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     5881 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     5941 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     6001 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     6061 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     6121 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     6181 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     6241 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     6301 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     6361 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     6421 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     6481 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     6541 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     6601 ggttgtaaca ctggcaaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6661 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6721 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6781 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6841 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6901 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6961 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7021 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7081 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacggttcc
     7141 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7201 gagggagctt ccagggggaa acgcctggta tctttatagt cctgt
//
