LOCUS       sequence-380740-        7275 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 7275)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7275)
  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-gcryBFP.2
FEATURES             Location/Qualifiers
     source          1..7275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(17..28,29..191)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    278..299
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(314..319,320..337,338..344)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    352..368
                     /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     357..379
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     376..392
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     376..392
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             388..738
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSLLDNGPI
                     TLNNANYLKLLS"
     promoter        413..431
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(479..497)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(507..524)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(507..523)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(516..538)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    698..729
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(735..768)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             945..1619
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             945..1619
                     /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(1072..1090)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1340..1359
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1534..1557
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3494..3514
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3518..4219
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4229..4453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4523..4604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4728..4809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4958..4991)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4997..5028
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5033..5066
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5246..5264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5287..5474,5475..5486)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5540..5559)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5551..6360
                     /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(5618..5637)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6503..7091
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6992..7011
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7245..7262
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttaaatggt accgagcaga ggtgtaaaaa gtactcaaaa attttactca agtgaaagta
       61 caagtactta gggaaaattt tactcaatta aaagtaaaag tatctggcta gaatcttact
      121 tgagtaaaag taaaaaagta ctccattaaa attgtacttg agtattaagg aagtaaaagt
      181 aaaagcaaga aagaaaacta gagtggtctc cctttagtga gggtcaattg atgggaggcg
      241 ttcgggccac agcggacacg caggtgcgca acgcaattaa tgtgagttag ctcactcatt
      301 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
      361 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
      421 ctcactaaag ggaacaaaag ctggaggacc tcgagggggg gcccggtacc caattcgccc
      481 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
      541 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
      601 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
      661 tggacctgcc acacttcctt gctagacaat gggcccataa ctttaaataa tgccaattat
      721 ttaaagttac tatcataact tcgtatagca tacattatac gaagttatac ctaggcttac
      781 cacccactag cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt
      841 cacatgctca ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct
      901 cattcattta cagtcagccg gaattcaccc gggtaccgga aggtgcctcc tccgaggacg
      961 tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt
     1021 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     1081 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttccagt
     1141 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     1201 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     1261 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     1321 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     1381 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg aggctgaagc
     1441 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacatggcc aagaagcccg
     1501 tgcagctgcc cggcgcctac aagaccgaca tcaagctgga catcacctcc cacaacgagg
     1561 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggagcctaag
     1621 actagtaatt aagtctcagc caccgttaac tgaacatgtc aaaacctgtg gagactgttg
     1681 agatttgatg ttctgaaaag ataaagccta taaataaaat gttgcccaaa tttcctgcct
     1741 gatgtttttc tttgtctttg ctacatggct ttgctgctcg gatcggctca ctctgtgtat
     1801 gccacgttca ctttgtactc tccttctcac ggtaggttta ttatttttag atgtgcagtt
     1861 agtttctgtg aaataacaca ccacacactg atattgtctg tgcattgact tggtgagtgc
     1921 acattgtttt tgatcttgac atatttatat ttgattgatc aggtgaactg tgtgaatcta
     1981 aagtgctcca tacagatgtt ctgcattgaa aatattctca ttttattagt ggaagtgagt
     2041 gtatgccaca tccaatcaat ttcagcaaac accccagtat gatttaatgc aaaaaaatga
     2101 aggtatcaaa cacgcattac tactttgcag ttaaatattt aacatttatt ccaacacgaa
     2161 aaaaagcagt aaataacact ttgacaaaca cgtcaggaca tcttattttt gtcaccctca
     2221 caggcaattt agtataatat attatatata tatatatatc atataataat attcagtata
     2281 atatatatat atatatcata ttataatatt cagtataata taaaacacaa acacatatat
     2341 gtataatata atataacatt tttatttatt gagatgcctc tatggaccgt gttataagaa
     2401 gtaaagatca ggagaagtaa acatgaagtg taattatgaa tactgatgtt aaattaagct
     2461 atgatgagtt ttcactgtta atttaccatc tcaattaaat gttgatgcct ccatgaccaa
     2521 gttaagcaga tgagactgag gacaactgta gaagacaaga tgttcacttt gctgaatata
     2581 gctggcttga cagttatcta tgactctata aatatatata tatttttttt tttataaaat
     2641 gatttattta taactatata tccatttctc agacaggtgc ttcatatccc tcactcccgt
     2701 agctgtccat gctggatctg tccccgttgt ttttaaaaag ctaaataagt tattaacatg
     2761 actgcatcca gcgagccaaa cctgtctggt gtacagctac cagagaagct tgagatccta
     2821 gtcaccggcg ttcctagttc tttaaactgt tacaaagtgt ttggaccagc aactgaattg
     2881 cactgtaaat taccacatca gggtctccgg ggtcaacgct gctaagcaat ggactggaaa
     2941 atttgtgaca ttgagttttt ggaataactg ggaatgctgg ataactcttt gggtaaagct
     3001 atggaacaaa agtaccagca tgaactaaga aatgactgtc acagcacaac atcaaacaca
     3061 atgcaagaca aaggttctct gcttttgcct tacttgacag aaaaagtaag ctctaggttc
     3121 atcgcatgca gacagcagca atacatgaca tatataaagg ctggctcttt tctcccttcc
     3181 attgaactga aacttccact cagtcagact tgcgggggat ccacagcctg actaactcga
     3241 caaacccccc caaacctaag gtgagttgat ctttaagctt tttacatttt cagctcgcat
     3301 atatcaattc gcgcgcattg tcacactagc acccactagc ggtcagactg cagattgcag
     3361 cacgaaacag gaagctgact ccacatggtc acatgctcac tgaagtgttg acttccctga
     3421 cagctgtgca ctttctaaac cggttttctc attcatttac agttcagccg gaattcacct
     3481 cgagaccatg gctccaaaga agaagcgtaa ggtaaccatg agcgagctga ttaaggagaa
     3541 catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc
     3601 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg
     3661 cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac
     3721 cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt
     3781 cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac
     3841 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc
     3901 caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta
     3961 ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag
     4021 ccatctgatc gcaaacatca agaccacata tagatccaag aaacccgcta agaacctcaa
     4081 gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga
     4141 gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact
     4201 ggggcacaag ctcaattaaa gctgagatct gtgccttcta gttgccagcc atctgttgtt
     4261 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     4321 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     4381 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     4441 gtgggctcta tggcttctga ggcggaaaga accaaagggc gaattaattc attgatgagt
     4501 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     4561 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     4621 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     4681 tctacaaatg tggtcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4741 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4801 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     4861 gaggtttttt aaagcaagta aaacctctac aaatgtggtc attcagatca attaaccctc
     4921 acccgggcta gcgatgcatg ggagctcaag acgtctgata acttcgtata gcatacatta
     4981 tacgaagtta tctacctaac tttaaataat gccaattatt taaagttact tccgcgcccg
     5041 gggagcccaa gggcacgccc tggcacctga cgtacgttgt ctagcaagga agtgaagagc
     5101 tatccggata tatatgtatg ttatcataac tgtactggct atgtcaggtg gtaatgactg
     5161 taacgttacg ttactcgttg taggcacgac attgaatatg ctcttctccg ctgtggcccg
     5221 aacgcctccc gctagcgatg ggatctaata cgactcacta tagggagacc aagggaaaat
     5281 agaatgaagt gatctccaaa aaataagtac tttttgactg taaataaaat tgtaaggagt
     5341 aaaaagtact tttttttcta aaaaaatgta attaagtaaa agtaaaagta ttgattttta
     5401 attgtactca agtaaagtaa aaatccccaa aaataatact taagtacagt aatcaagtaa
     5461 aattactcaa gtactttaca cctctgggat ccagctgcat catgaacaat aaaactgtct
     5521 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg
     5581 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     5641 gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg
     5701 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     5761 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     5821 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa
     5881 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     5941 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     6001 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     6061 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     6121 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     6181 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     6241 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     6301 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     6361 tcagaattgg ttaattggtt gtaacactgg caaaaaggat ctaggtgaag atcctttttg
     6421 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6481 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6541 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6601 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6661 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6721 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6781 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6841 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6901 aaagcgccac ggttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6961 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7021 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7081 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7141 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     7201 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     7261 aggaagcgga acaga
//
