LOCUS       sequence-381108-        7313 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 7313)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7313)
  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-gcryBFP.1
FEATURES             Location/Qualifiers
     source          1..7313
                     /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             946..1002
                     /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             1009..1446
                     /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             3532..3552
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3556..4257
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4267..4491
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4561..4642
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4766..4847
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4996..5029)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5035..5066
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5071..5104
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5284..5302
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5325..5512,5513..5524)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5578..5597)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5589..6398
                     /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(5656..5675)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6541..7129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7030..7049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7283..7300
                     /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 cagttcagcc ggaattcacc cgggtaccgg aaggtgccac gaacttctct
      961 ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc cttctagaaa gctactgtct
     1021 tctatcgaac aagcatgcga tatttgccga cttaaaaagc tcaagtgctc caaagaaaaa
     1081 ccgaagtgcg ccaagtgtct gaagaacaac tgggagtgtc gctactctcc caaaaccaaa
     1141 aggtctccgc tgactagggc acatctgaca gaagtggaat caaggctaga aagactggaa
     1201 cagctatttc tactgatttt tcctcgagaa gaccttgaca tgattttgaa aatggattct
     1261 ttacaggata taaaagcatt gttaacagga ttatttgtac aagataatgt gaataaagat
     1321 gccgtcacag atagattggc ttcagtggag actgatatgc ctctaacatt gagacagcat
     1381 agaataagtg cgacatcatc atcggaagag agtagtaaca aaggtcaaag acagttgact
     1441 gtatcgtcga ggtcgacccc gggaatacag atctctcgag ccgccccccc gaccgatgtc
     1501 agcctggggg acgagctcca cttagacggc gaggacgtgg cgatggcgca tgccgacgcg
     1561 ctagacgatt tcgatctgga catgttgggg gacggggatt ccccgggtcc gggatttacc
     1621 ccccacgact ccgcccccta cggcgctctg gatatgtaac tagtaattaa gtctcagcca
     1681 ccgttaactg aacatgtcaa aacctgtgga gactgttgag atttgatgtt ctgaaaagat
     1741 aaagcctata aataaaatgt tgcccaaatt tcctgcctga tgtttttctt tgtctttgct
     1801 acatggcttt gctgctcgga tcggctcact ctgtgtatgc cacgttcact ttgtactctc
     1861 cttctcacgg taggtttatt atttttagat gtgcagttag tttctgtgaa ataacacacc
     1921 acacactgat attgtctgtg cattgacttg gtgagtgcac attgtttttg atcttgacat
     1981 atttatattt gattgatcag gtgaactgtg tgaatctaaa gtgctccata cagatgttct
     2041 gcattgaaaa tattctcatt ttattagtgg aagtgagtgt atgccacatc caatcaattt
     2101 cagcaaacac cccagtatga tttaatgcaa aaaaatgaag gtatcaaaca cgcattacta
     2161 ctttgcagtt aaatatttaa catttattcc aacacgaaaa aaagcagtaa ataacacttt
     2221 gacaaacacg tcaggacatc ttatttttgt caccctcaca ggcaatttag tataatatat
     2281 tatatatata tatatatcat ataataatat tcagtataat atatatatat atatcatatt
     2341 ataatattca gtataatata aaacacaaac acatatatgt ataatataat ataacatttt
     2401 tatttattga gatgcctcta tggaccgtgt tataagaagt aaagatcagg agaagtaaac
     2461 atgaagtgta attatgaata ctgatgttaa attaagctat gatgagtttt cactgttaat
     2521 ttaccatctc aattaaatgt tgatgcctcc atgaccaagt taagcagatg agactgagga
     2581 caactgtaga agacaagatg ttcactttgc tgaatatagc tggcttgaca gttatctatg
     2641 actctataaa tatatatata tttttttttt tataaaatga tttatttata actatatatc
     2701 catttctcag acaggtgctt catatccctc actcccgtag ctgtccatgc tggatctgtc
     2761 cccgttgttt ttaaaaagct aaataagtta ttaacatgac tgcatccagc gagccaaacc
     2821 tgtctggtgt acagctacca gagaagcttg agatcctagt caccggcgtt cctagttctt
     2881 taaactgtta caaagtgttt ggaccagcaa ctgaattgca ctgtaaatta ccacatcagg
     2941 gtctccgggg tcaacgctgc taagcaatgg actggaaaat ttgtgacatt gagtttttgg
     3001 aataactggg aatgctggat aactctttgg gtaaagctat ggaacaaaag taccagcatg
     3061 aactaagaaa tgactgtcac agcacaacat caaacacaat gcaagacaaa ggttctctgc
     3121 ttttgcctta cttgacagaa aaagtaagct ctaggttcat cgcatgcaga cagcagcaat
     3181 acatgacata tataaaggct ggctcttttc tcccttccat tgaactgaaa cttccactca
     3241 gtcagacttg cgggggatcc acagcctgac taactcgaca aaccccccca aacctaaggt
     3301 gagttgatct ttaagctttt tacattttca gctcgcatat atcaattcgc gcgcattgtc
     3361 acactagcac ccactagcgg tcagactgca gattgcagca cgaaacagga agctgactcc
     3421 acatggtcac atgctcactg aagtgttgac ttccctgaca gctgtgcact ttctaaaccg
     3481 gttttctcat tcatttacag ttcagccgga attcacctcg agaccatggc tccaaagaag
     3541 aagcgtaagg taaccatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
     3601 gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac
     3661 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
     3721 gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc
     3781 atccccgact tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca
     3841 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
     3901 atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag
     3961 aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa
     4021 ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag
     4081 accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg
     4141 gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag
     4201 gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct caattaaagc
     4261 tgagatctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4321 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4381 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4441 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     4501 cggaaagaac caaagggcga attaattcat tgatgagttt ggacaaacca caactagaat
     4561 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4621 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4681 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattgatg
     4741 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4801 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4861 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     4921 acctctacaa atgtggtcat tcagatcaat taaccctcac ccgggctagc gatgcatggg
     4981 agctcaagac gtctgataac ttcgtatagc atacattata cgaagttatc tacctaactt
     5041 taaataatgc caattattta aagttacttc cgcgcccggg gagcccaagg gcacgccctg
     5101 gcacctgacg tacgttgtct agcaaggaag tgaagagcta tccggatata tatgtatgtt
     5161 atcataactg tactggctat gtcaggtggt aatgactgta acgttacgtt actcgttgta
     5221 ggcacgacat tgaatatgct cttctccgct gtggcccgaa cgcctcccgc tagcgatggg
     5281 atctaatacg actcactata gggagaccaa gggaaaatag aatgaagtga tctccaaaaa
     5341 ataagtactt tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa
     5401 aaaatgtaat taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa
     5461 atccccaaaa ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc
     5521 tctgggatcc agctgcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     5581 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     5641 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     5701 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     5761 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     5821 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     5881 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     5941 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     6001 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     6061 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     6121 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     6181 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     6241 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     6301 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     6361 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     6421 aacactggca aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6481 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6541 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6601 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6661 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6721 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6781 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6841 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6901 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgg ttcccgaagg
     6961 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7021 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7081 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7141 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     7201 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     7261 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaac aga
//
