LOCUS       sequence-381106-        7266 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 7266)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7266)
  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.3
FEATURES             Location/Qualifiers
     source          1..7266
                     /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"
     CDS             3739..3759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3763..4464
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4474..4698
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4768..4849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4973..5054
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5203..5236)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5242..5273
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5278..5311
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5491..5509
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5532..5719,5720..5731)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5785..5804)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5796..6605
                     /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(5863..5882)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6748..7266,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7237..7256
                     /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 cacctcgaga ccatggctcc aaagaagaag cgtaaggtaa ccatgagcga gctgattaag
     3781 gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc
     3841 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     3901 gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc
     3961 aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag
     4021 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     4081 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     4141 acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg
     4201 ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc
     4261 gggagccatc tgatcgcaaa catcaagacc acatatagat ccaagaaacc cgctaagaac
     4321 ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac
     4381 aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc
     4441 aaactggggc acaagctcaa ttaaagctga gatctgtgcc ttctagttgc cagccatctg
     4501 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     4561 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     4621 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     4681 atgcggtggg ctctatggct tctgaggcgg aaagaaccaa agggcgaatt aattcattga
     4741 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4801 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     4861 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     4921 aaacctctac aaatgtggtc attgatgagt ttggacaaac cacaactaga atgcagtgaa
     4981 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     5041 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     5101 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattca gatcaattaa
     5161 ccctcacccg ggctagcgat gcatgggagc tcaagacgtc tgataacttc gtatagcata
     5221 cattatacga agttatctac ctaactttaa ataatgccaa ttatttaaag ttacttccgc
     5281 gcccggggag cccaagggca cgccctggca cctgacgtac gttgtctagc aaggaagtga
     5341 agagctatcc ggatatatat gtatgttatc ataactgtac tggctatgtc aggtggtaat
     5401 gactgtaacg ttacgttact cgttgtaggc acgacattga atatgctctt ctccgctgtg
     5461 gcccgaacgc ctcccgctag cgatgggatc taatacgact cactataggg agaccaaggg
     5521 aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa
     5581 ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat
     5641 ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca
     5701 agtaaaatta ctcaagtact ttacacctct gggatccagc tgcatcatga acaataaaac
     5761 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     5821 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     5881 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     5941 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     6001 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     6061 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     6121 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     6181 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     6241 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     6301 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     6361 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     6421 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     6481 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     6541 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     6601 tctaatcaga attggttaat tggttgtaac actggcaaaa aggatctagg tgaagatcct
     6661 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6721 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6781 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6841 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6901 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6961 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7021 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7081 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7141 atgagaaagc gccacggttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7201 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7261 tcctgt
//
