LOCUS       sequence-381113-        7287 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 7287)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7287)
  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-insGFP.1
FEATURES             Location/Qualifiers
     source          1..7287
                     /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             1012..1449
                     /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"
     polyA_signal    4241..4465
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4535..4616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4740..4821
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4970..5003)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5009..5040
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5045..5078
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5258..5276
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5299..5486,5487..5498)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5552..5571)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5563..6372
                     /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(5630..5649)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6515..7103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7004..7023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7257..7274
                     /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 ctacgtacca gaagctactg
     1021 tcttctatcg aacaagcatg cgatatttgc cgacttaaaa agctcaagtg ctccaaagaa
     1081 aaaccgaagt gcgccaagtg tctgaagaac aactgggagt gtcgctactc tcccaaaacc
     1141 aaaaggtctc cgctgactag ggcacatctg acagaagtgg aatcaaggct agaaagactg
     1201 gaacagctat ttctactgat ttttcctcga gaagaccttg acatgatttt gaaaatggat
     1261 tctttacagg atataaaagc attgttaaca ggattatttg tacaagataa tgtgaataaa
     1321 gatgccgtca cagatagatt ggcttcagtg gagactgata tgcctctaac attgagacag
     1381 catagaataa gtgcgacatc atcatcggaa gagagtagta acaaaggtca aagacagttg
     1441 actgtatcgt cgaggtcgac cccgggaata cagatctctc gagccgcccc cccgaccgat
     1501 gtcagcctgg gggacgagct ccacttagac ggcgaggacg tggcgatggc gcatgccgac
     1561 gcgctagacg atttcgatct ggacatgttg ggggacgggg attccccggg tccgggattt
     1621 accccccacg actccgcccc ctacggcgct ctggatatgt aactagtaat taagtctcag
     1681 ccaccgttaa ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa
     1741 gataaagcct ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt
     1801 gctacatggc tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact
     1861 ctccttctca cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac
     1921 accacacact gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga
     1981 catatttata tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt
     2041 tctgcattga aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa
     2101 tttcagcaaa caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta
     2161 ctactttgca gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac
     2221 tttgacaaac acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata
     2281 tattatatat atatatatat catataataa tattcagtat aatatatata tatatatcat
     2341 attataatat tcagtataat ataaaacaca aacacatata tgtataatat aatataacat
     2401 ttttatttat tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta
     2461 aacatgaagt gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt
     2521 aatttaccat ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga
     2581 ggacaactgt agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct
     2641 atgactctat aaatatatat atattttttt ttttataaaa tgatttattt ataactatat
     2701 atccatttct cagacaggtg cttcatatcc ctcactcccg tagctgtcca tgctggatct
     2761 gtccccgttg tttttaaaaa gctaaataag ttattaacat gactgcatcc agcgagccaa
     2821 acctgtctgg tgtacagcta ccagagaagc catatgggcg cacaaaatga tctaatagta
     2881 tactataaaa tctttcaaaa tccaggccac taccctatga ctaaaaccac acatattagg
     2941 ttttaaaaaa aagtcacaat gaagacgtca caaacgttcc tgaagactaa tgactcgtct
     3001 aaccccatct taccttctgc tgtttcactc aaagaaaagc caccagctgt ctctgtgatc
     3061 tgctcattct gtgtaaactt gtgttctcaa caattaacaa ttaaccaaaa gcctctggag
     3121 ctgcgtgagc atcacgatgt gtttgtcttc tgtgagcgtg gcagtgtcca cgaagtcaat
     3181 gtgaattggt tggccaaaca cagctaatgt ggcatgtgtg attggacagc cacttaaaag
     3241 tgttaaagtg tgtttacgca caggattggt tttccgctga atcaggcaaa accagcagaa
     3301 ccgatctaaa ctcgagtata aaagtgagta ggagatgcta ttcgtcccaa aacatctcca
     3361 ccaccatatc caccattcct cgcctctgct tcgagaacag gtgagtgtcg agcgggatgg
     3421 taaatctaca gagaatgcgg agtgtagctt gtgtacatgt ttttgattaa cagagattgt
     3481 atgtgtgtgt ttgtgtcagt gtggaattcc tcgagaccat gagcggaggt gaggagctgt
     3541 tcgccggcat cgtgcccgtg ctgatcgagc tggacggcga cgtgcacggc cacaagttca
     3601 gcgtgcgcgg cgagggcgag ggcgacgccg actacggcaa gctggagatc aagttcatct
     3661 gcaccaccgg caagctgccc gtgccctggc ccaccctggt gaccaccctc tgctacggca
     3721 tccagtgctt cgcccgctac cccgagcaca tgaagatgaa cgacttcttc aagagcgcca
     3781 tgcccgaggg ctacatccag gagcgcacca tccagttcca ggacgacggc aagtacaaga
     3841 cccgcggcga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3901 aggacttcaa ggaggacggc aacatcctgg gccacaagct ggagtacagc ttcaacagcc
     3961 acaacgtgta catccgcccc gacaaggcca acaacggcct ggaggctaac ttcaagaccc
     4021 gccacaacat cgagggcggc ggcgtgcagc tggccgacca ctaccagacc aacgtgcccc
     4081 tgggcgacgg ccccgtgctg atccccatca accactacct gagcactcag accaagatca
     4141 gcaaggaccg caacgaggcc cgcgaccaca tggtgctcct ggagtccttc agcgcctgct
     4201 gccacaccca cggcatggac gagctgtata ggtaatagat ctgtgccttc tagttgccag
     4261 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4321 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4381 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4441 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccaaagg gcgaattaat
     4501 tcattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     4561 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     4621 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     4681 gcaagtaaaa cctctacaaa tgtggtcatt gatgagtttg gacaaaccac aactagaatg
     4741 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     4801 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     4861 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tcattcagat
     4921 caattaaccc tcacccgggc tagcgatgca tgggagctca agacgtctga taacttcgta
     4981 tagcatacat tatacgaagt tatctaccta actttaaata atgccaatta tttaaagtta
     5041 cttccgcgcc cggggagccc aagggcacgc cctggcacct gacgtacgtt gtctagcaag
     5101 gaagtgaaga gctatccgga tatatatgta tgttatcata actgtactgg ctatgtcagg
     5161 tggtaatgac tgtaacgtta cgttactcgt tgtaggcacg acattgaata tgctcttctc
     5221 cgctgtggcc cgaacgcctc ccgctagcga tgggatctaa tacgactcac tatagggaga
     5281 ccaagggaaa atagaatgaa gtgatctcca aaaaataagt actttttgac tgtaaataaa
     5341 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag
     5401 tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca
     5461 gtaatcaagt aaaattactc aagtacttta cacctctggg atccagctgc atcatgaaca
     5521 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     5581 gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     5641 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat
     5701 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     5761 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     5821 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag
     5881 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     5941 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     6001 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     6061 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     6121 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     6181 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     6241 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     6301 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     6361 gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcaaaaagg atctaggtga
     6421 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6481 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6541 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6601 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6661 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6901 gtgagctatg agaaagcgcc acggttcccg aagggagaaa ggcggacagg tatccggtaa
     6961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7021 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7081 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7141 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     7201 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     7261 agtcagtgag cgaggaagcg gaacaga
//
