LOCUS       sequence-381045-        7312 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 7312)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7312)
  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.2
FEATURES             Location/Qualifiers
     source          1..7312
                     /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..1001
                     /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             1008..1445
                     /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             3531..3551
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3555..4256
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4266..4490
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4560..4641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4765..4846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4995..5028)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5034..5065
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5070..5103
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5283..5301
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5324..5511,5512..5523)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5577..5596)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5588..6397
                     /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(5655..5674)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6540..7128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7029..7048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7282..7299
                     /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 aggtgccacg aacttctctc
      961 tgttaaagca agcaggagac gtggaagaaa accccggtcc ttctagaaag ctactgtctt
     1021 ctatcgaaca agcatgcgat atttgccgac ttaaaaagct caagtgctcc aaagaaaaac
     1081 cgaagtgcgc caagtgtctg aagaacaact gggagtgtcg ctactctccc aaaaccaaaa
     1141 ggtctccgct gactagggca catctgacag aagtggaatc aaggctagaa agactggaac
     1201 agctatttct actgattttt cctcgagaag accttgacat gattttgaaa atggattctt
     1261 tacaggatat aaaagcattg ttaacaggat tatttgtaca agataatgtg aataaagatg
     1321 ccgtcacaga tagattggct tcagtggaga ctgatatgcc tctaacattg agacagcata
     1381 gaataagtgc gacatcatca tcggaagaga gtagtaacaa aggtcaaaga cagttgactg
     1441 tatcgtcgag gtcgaccccg ggaatacaga tctctcgagc cgcccccccg accgatgtca
     1501 gcctggggga cgagctccac ttagacggcg aggacgtggc gatggcgcat gccgacgcgc
     1561 tagacgattt cgatctggac atgttggggg acggggattc cccgggtccg ggatttaccc
     1621 cccacgactc cgccccctac ggcgctctgg atatgtaact agtaattaag tctcagccac
     1681 cgttaactga acatgtcaaa acctgtggag actgttgaga tttgatgttc tgaaaagata
     1741 aagcctataa ataaaatgtt gcccaaattt cctgcctgat gtttttcttt gtctttgcta
     1801 catggctttg ctgctcggat cggctcactc tgtgtatgcc acgttcactt tgtactctcc
     1861 ttctcacggt aggtttatta tttttagatg tgcagttagt ttctgtgaaa taacacacca
     1921 cacactgata ttgtctgtgc attgacttgg tgagtgcaca ttgtttttga tcttgacata
     1981 tttatatttg attgatcagg tgaactgtgt gaatctaaag tgctccatac agatgttctg
     2041 cattgaaaat attctcattt tattagtgga agtgagtgta tgccacatcc aatcaatttc
     2101 agcaaacacc ccagtatgat ttaatgcaaa aaaatgaagg tatcaaacac gcattactac
     2161 tttgcagtta aatatttaac atttattcca acacgaaaaa aagcagtaaa taacactttg
     2221 acaaacacgt caggacatct tatttttgtc accctcacag gcaatttagt ataatatatt
     2281 atatatatat atatatcata taataatatt cagtataata tatatatata tatcatatta
     2341 taatattcag tataatataa aacacaaaca catatatgta taatataata taacattttt
     2401 atttattgag atgcctctat ggaccgtgtt ataagaagta aagatcagga gaagtaaaca
     2461 tgaagtgtaa ttatgaatac tgatgttaaa ttaagctatg atgagttttc actgttaatt
     2521 taccatctca attaaatgtt gatgcctcca tgaccaagtt aagcagatga gactgaggac
     2581 aactgtagaa gacaagatgt tcactttgct gaatatagct ggcttgacag ttatctatga
     2641 ctctataaat atatatatat tttttttttt ataaaatgat ttatttataa ctatatatcc
     2701 atttctcaga caggtgcttc atatccctca ctcccgtagc tgtccatgct ggatctgtcc
     2761 ccgttgtttt taaaaagcta aataagttat taacatgact gcatccagcg agccaaacct
     2821 gtctggtgta cagctaccag agaagcttga gatcctagtc accggcgttc ctagttcttt
     2881 aaactgttac aaagtgtttg gaccagcaac tgaattgcac tgtaaattac cacatcaggg
     2941 tctccggggt caacgctgct aagcaatgga ctggaaaatt tgtgacattg agtttttgga
     3001 ataactggga atgctggata actctttggg taaagctatg gaacaaaagt accagcatga
     3061 actaagaaat gactgtcaca gcacaacatc aaacacaatg caagacaaag gttctctgct
     3121 tttgccttac ttgacagaaa aagtaagctc taggttcatc gcatgcagac agcagcaata
     3181 catgacatat ataaaggctg gctcttttct cccttccatt gaactgaaac ttccactcag
     3241 tcagacttgc gggggatcca cagcctgact aactcgacaa acccccccaa acctaaggtg
     3301 agttgatctt taagcttttt acattttcag ctcgcatata tcaattcgcg cgcattgtca
     3361 cactagcacc cactagcggt cagactgcag attgcagcac gaaacaggaa gctgactcca
     3421 catggtcaca tgctcactga agtgttgact tccctgacag ctgtgcactt tctaaaccgg
     3481 ttttctcatt catttacagt tcagccggaa ttcacctcga gaccatggct ccaaagaaga
     3541 agcgtaaggt aaccatgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg
     3601 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     3661 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     3721 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     3781 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     3841 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     3901 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     3961 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     4021 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga
     4081 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     4141 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     4201 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctc aattaaagct
     4261 gagatctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4321 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4381 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4441 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     4501 ggaaagaacc aaagggcgaa ttaattcatt gatgagtttg gacaaaccac aactagaatg
     4561 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     4621 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     4681 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tcattgatga
     4741 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4801 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     4861 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     4921 cctctacaaa tgtggtcatt cagatcaatt aaccctcacc cgggctagcg atgcatggga
     4981 gctcaagacg tctgataact tcgtatagca tacattatac gaagttatct acctaacttt
     5041 aaataatgcc aattatttaa agttacttcc gcgcccgggg agcccaaggg cacgccctgg
     5101 cacctgacgt acgttgtcta gcaaggaagt gaagagctat ccggatatat atgtatgtta
     5161 tcataactgt actggctatg tcaggtggta atgactgtaa cgttacgtta ctcgttgtag
     5221 gcacgacatt gaatatgctc ttctccgctg tggcccgaac gcctcccgct agcgatggga
     5281 tctaatacga ctcactatag ggagaccaag ggaaaataga atgaagtgat ctccaaaaaa
     5341 taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa
     5401 aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa
     5461 tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct
     5521 ctgggatcca gctgcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     5581 gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     5641 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     5701 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     5761 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     5821 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     5881 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     5941 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     6001 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     6061 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     6121 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     6181 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     6241 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     6301 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     6361 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     6421 acactggcaa aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6481 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6541 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6601 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6661 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6721 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6781 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6841 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6901 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacggt tcccgaaggg
     6961 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7021 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7081 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7141 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7201 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7261 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaca ga
//
