LOCUS       sequence-381141-        7324 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 7324)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7324)
  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-2aG4-gcryGFP.3
FEATURES             Location/Qualifiers
     source          1..7324
                     /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             944..1000
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1007..1444
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3530..3550
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3554..3556,3557..3559,3560..4273)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3554..3556,3557..3559,3560..4270)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3599..3620)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3860..3879)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4207..4228
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4278..4502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4572..4653
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4777..4858
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5007..5040)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5046..5077
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5082..5115
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5295..5313
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5336..5523,5524..5535)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5589..5608)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5600..6409
                     /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(5667..5686)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6552..7140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7041..7060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7294..7311
                     /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 cagcagccgg aattcacccg ggtaccggaa ggtgccacga acttctctct
      961 gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct tctagaaagc tactgtcttc
     1021 tatcgaacaa gcatgcgata tttgccgact taaaaagctc aagtgctcca aagaaaaacc
     1081 gaagtgcgcc aagtgtctga agaacaactg ggagtgtcgc tactctccca aaaccaaaag
     1141 gtctccgctg actagggcac atctgacaga agtggaatca aggctagaaa gactggaaca
     1201 gctatttcta ctgatttttc ctcgagaaga ccttgacatg attttgaaaa tggattcttt
     1261 acaggatata aaagcattgt taacaggatt atttgtacaa gataatgtga ataaagatgc
     1321 cgtcacagat agattggctt cagtggagac tgatatgcct ctaacattga gacagcatag
     1381 aataagtgcg acatcatcat cggaagagag tagtaacaaa ggtcaaagac agttgactgt
     1441 atcgtcgagg tcgaccccgg gaatacagat ctctcgagcc gcccccccga ccgatgtcag
     1501 cctgggggac gagctccact tagacggcga ggacgtggcg atggcgcatg ccgacgcgct
     1561 agacgatttc gatctggaca tgttggggga cggggattcc ccgggtccgg gatttacccc
     1621 ccacgactcc gccccctacg gcgctctgga tatgtaacta gtaattaagt ctcagccacc
     1681 gttaactgaa catgtcaaaa cctgtggaga ctgttgagat ttgatgttct gaaaagataa
     1741 agcctataaa taaaatgttg cccaaatttc ctgcctgatg tttttctttg tctttgctac
     1801 atggctttgc tgctcggatc ggctcactct gtgtatgcca cgttcacttt gtactctcct
     1861 tctcacggta ggtttattat ttttagatgt gcagttagtt tctgtgaaat aacacaccac
     1921 acactgatat tgtctgtgca ttgacttggt gagtgcacat tgtttttgat cttgacatat
     1981 ttatatttga ttgatcaggt gaactgtgtg aatctaaagt gctccataca gatgttctgc
     2041 attgaaaata ttctcatttt attagtggaa gtgagtgtat gccacatcca atcaatttca
     2101 gcaaacaccc cagtatgatt taatgcaaaa aaatgaaggt atcaaacacg cattactact
     2161 ttgcagttaa atatttaaca tttattccaa cacgaaaaaa agcagtaaat aacactttga
     2221 caaacacgtc aggacatctt atttttgtca ccctcacagg caatttagta taatatatta
     2281 tatatatata tatatcatat aataatattc agtataatat atatatatat atcatattat
     2341 aatattcagt ataatataaa acacaaacac atatatgtat aatataatat aacattttta
     2401 tttattgaga tgcctctatg gaccgtgtta taagaagtaa agatcaggag aagtaaacat
     2461 gaagtgtaat tatgaatact gatgttaaat taagctatga tgagttttca ctgttaattt
     2521 accatctcaa ttaaatgttg atgcctccat gaccaagtta agcagatgag actgaggaca
     2581 actgtagaag acaagatgtt cactttgctg aatatagctg gcttgacagt tatctatgac
     2641 tctataaata tatatatatt ttttttttta taaaatgatt tatttataac tatatatcca
     2701 tttctcagac aggtgcttca tatccctcac tcccgtagct gtccatgctg gatctgtccc
     2761 cgttgttttt aaaaagctaa ataagttatt aacatgactg catccagcga gccaaacctg
     2821 tctggtgtac agctaccaga gaagcttgag atcctagtca ccggcgttcc tagttcttta
     2881 aactgttaca aagtgtttgg accagcaact gaattgcact gtaaattacc acatcagggt
     2941 ctccggggtc aacgctgcta agcaatggac tggaaaattt gtgacattga gtttttggaa
     3001 taactgggaa tgctggataa ctctttgggt aaagctatgg aacaaaagta ccagcatgaa
     3061 ctaagaaatg actgtcacag cacaacatca aacacaatgc aagacaaagg ttctctgctt
     3121 ttgccttact tgacagaaaa agtaagctct aggttcatcg catgcagaca gcagcaatac
     3181 atgacatata taaaggctgg ctcttttctc ccttccattg aactgaaact tccactcagt
     3241 cagacttgcg ggggatccac agcctgacta actcgacaaa cccccccaaa cctaaggtga
     3301 gttgatcttt aagcttttta cattttcagc tcgcatatat caattcgcgc gcattgtcac
     3361 actagcaccc actagcggtc agactgcaga ttgcagcacg aaacaggaag ctgactccac
     3421 atggtcacat gctcactgaa gtgttgactt ccctgacagc tgtgcacttt ctaaaccggt
     3481 tttctcattc atttacagtt cagccggaat tcacctcgag accatggctc caaagaagaa
     3541 gcgtaaggta accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     3601 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3661 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3721 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     3781 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3841 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3901 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3961 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     4021 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     4081 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     4141 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     4201 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     4261 gctgtacaag taaagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4321 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4381 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4441 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4501 ggcttctgag gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc
     4561 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4621 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4681 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4741 ggtcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4801 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4861 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4921 aagcaagtaa aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag
     4981 cgatgcatgg gagctcaaga cgtctgataa cttcgtatag catacattat acgaagttat
     5041 ctacctaact ttaaataatg ccaattattt aaagttactt ccgcgcccgg ggagcccaag
     5101 ggcacgccct ggcacctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat
     5161 atatgtatgt tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt
     5221 tactcgttgt aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg
     5281 ctagcgatgg gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg
     5341 atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt
     5401 ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa
     5461 gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag
     5521 tactttacac ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa
     5581 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     5641 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     5701 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     5761 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     5821 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     5881 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     5941 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     6001 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     6061 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     6121 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     6181 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     6241 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     6301 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     6361 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     6421 taattggttg taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg
     6481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6661 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6901 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6961 gttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7201 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7261 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7321 caga
//
