LOCUS       sequence-381104-        7180 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 7180)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7180)
  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-cmlc2GFP.2
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     224..241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(271..282,283..445)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    532..553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(568..573,574..591,592..598)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    606..622
                     /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     611..633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     630..646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             642..992
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSLLDNGPI
                     TLNNANYLKLLS"
     promoter        667..685
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(733..751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(761..778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(761..777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(770..792)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    952..983
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(989..1022)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1199..1255
                     /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             1262..1699
                     /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    4388..4612
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4682..4763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4887..4968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5117..5150)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5156..5187
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5192..5225
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5405..5423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5446..5633,5634..5645)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5699..5718)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5710..6519
                     /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(5777..5796)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6662..7180,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7151..7170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa ttaatgtgag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg
      721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
      781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      901 gcctgaatgg cgaatggacc tgccacactt ccttgctaga caatgggccc ataactttaa
      961 ataatgccaa ttatttaaag ttactatcat aacttcgtat agcatacatt atacgaagtt
     1021 atacctaggc ttaccaccca ctagcggtca gactgcagat tgcagcacga aacaggaagc
     1081 tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct gtgcactttc
     1141 taaaccggtt ttctcattca tttacagtca gccggaattc acccgggtac cggaaggtgc
     1201 cacgaacttc tctctgttaa agcaagcagg agacgtggaa gaaaaccccg gtccttctag
     1261 aaagctactg tcttctatcg aacaagcatg cgatatttgc cgacttaaaa agctcaagtg
     1321 ctccaaagaa aaaccgaagt gcgccaagtg tctgaagaac aactgggagt gtcgctactc
     1381 tcccaaaacc aaaaggtctc cgctgactag ggcacatctg acagaagtgg aatcaaggct
     1441 agaaagactg gaacagctat ttctactgat ttttcctcga gaagaccttg acatgatttt
     1501 gaaaatggat tctttacagg atataaaagc attgttaaca ggattatttg tacaagataa
     1561 tgtgaataaa gatgccgtca cagatagatt ggcttcagtg gagactgata tgcctctaac
     1621 attgagacag catagaataa gtgcgacatc atcatcggaa gagagtagta acaaaggtca
     1681 aagacagttg actgtatcgt cgaggtcgac cccgggaata cagatctctc gagccgcccc
     1741 cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg tggcgatggc
     1801 gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg attccccggg
     1861 tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgt aactagtaat
     1921 taagtctcag ccaccgttaa ctgaacatgt caaaacctgt ggagactgtt gagatttgat
     1981 gttctgaaaa gataaagcct ataaataaaa tgttgcccaa atttcctgcc tgatgttttt
     2041 ctttgtcttt gctacatggc tttgctgctc ggatcggctc actctgtgta tgccacgttc
     2101 actttgtact ctccttctca cggtaggttt attattttta gatgtgcagt tagtttctgt
     2161 gaaataacac accacacact gatattgtct gtgcattgac ttggtgagtg cacattgttt
     2221 ttgatcttga catatttata tttgattgat caggtgaact gtgtgaatct aaagtgctcc
     2281 atacagatgt tctgcattga aaatattctc attttattag tggaagtgag tgtatgccac
     2341 atccaatcaa tttcagcaaa caccccagta tgatttaatg caaaaaaatg aaggtatcaa
     2401 acacgcatta ctactttgca gttaaatatt taacatttat tccaacacga aaaaaagcag
     2461 taaataacac tttgacaaac acgtcaggac atcttatttt tgtcaccctc acaggcaatt
     2521 tagtataata tattatatat atatatatat catataataa tattcagtat aatatatata
     2581 tatatatcat attataatat tcagtataat ataaaacaca aacacatata tgtataatat
     2641 aatataacat ttttatttat tgagatgcct ctatggaccg tgttataaga agtaaagatc
     2701 aggagaagta aacatgaagt gtaattatga atactgatgt taaattaagc tatgatgagt
     2761 tttcactgtt aatttaccat ctcaattaaa tgttgatgcc tccatgacca agttaagcag
     2821 atgagactga ggacaactgt agaagacaag atgttcactt tgctgaatat agctggcttg
     2881 acagttatct atgactctat aaatatatat atattttttt ttttataaaa tgatttattt
     2941 ataactatat atccatttct cagacaggtg cttcatatcc ctcactcccg tagctgtcca
     3001 tgctggatct gtccccgttg tttttaaaaa gctaaataag ttattaacat gactgcatcc
     3061 agcgagccaa acctgtctgg tgtacagcta ccagagaagc ttgagatcct agtcaccggc
     3121 ggcattcatc catccttttc atccctcaaa tctctcattc acgtccccct ccccatctgc
     3181 acactttatc tcattttcca ccctgctgga atctgagcac ttgtgcagtt atcagggctc
     3241 ctgtatttag gaggctctgg gtgtccatgt aggggacgaa cagaaacact gcagaccttt
     3301 atagaagaac aaatgataag agtcctcata cataaagact ccattagaaa cgtcagtgac
     3361 ccaggagccc agaccaacag caaagcagac agtgaaggat ccacagcctg actaactcga
     3421 caaacccccc caaacctaag gtgagttgat ctttaagctt tttacatttt cagctcgcat
     3481 atatcaattc gcgcgcattg tcacactagc acccactagc ggtcagactg cagattgcag
     3541 cacgaaacag gaagctgact ccacatggtc acatgctcac tgaagtgttg acttccctga
     3601 cagctgtgca ctttctaaac cggttttctc attcatttac agttcagccg gaattcctcg
     3661 agaccatgag cggaggtgag gagctgttcg ccggcatcgt gcccgtgctg atcgagctgg
     3721 acggcgacgt gcacggccac aagttcagcg tgcgcggcga gggcgagggc gacgccgact
     3781 acggcaagct ggagatcaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     3841 ccctggtgac caccctctgc tacggcatcc agtgcttcgc ccgctacccc gagcacatga
     3901 agatgaacga cttcttcaag agcgccatgc ccgagggcta catccaggag cgcaccatcc
     3961 agttccagga cgacggcaag tacaagaccc gcggcgaggt gaagttcgag ggcgacaccc
     4021 tggtgaaccg catcgagctg aagggcaagg acttcaagga ggacggcaac atcctgggcc
     4081 acaagctgga gtacagcttc aacagccaca acgtgtacat ccgccccgac aaggccaaca
     4141 acggcctgga ggctaacttc aagacccgcc acaacatcga gggcggcggc gtgcagctgg
     4201 ccgaccacta ccagaccaac gtgcccctgg gcgacggccc cgtgctgatc cccatcaacc
     4261 actacctgag cactcagacc aagatcagca aggaccgcaa cgaggcccgc gaccacatgg
     4321 tgctcctgga gtccttcagc gcctgctgcc acacccacgg catggacgag ctgtataggt
     4381 aatagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4441 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4501 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4561 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4621 gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc acaactagaa
     4681 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     4741 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     4801 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattgat
     4861 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4921 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4981 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     5041 aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag cgatgcatgg
     5101 gagctcaaga cgtctgataa cttcgtatag catacattat acgaagttat ctacctaact
     5161 ttaaataatg ccaattattt aaagttactt ccgcgcccgg ggagcccaag ggcacgccct
     5221 ggcacctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat atatgtatgt
     5281 tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt tactcgttgt
     5341 aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg ctagcgatgg
     5401 gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg atctccaaaa
     5461 aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt ttttttctaa
     5521 aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa
     5581 aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag tactttacac
     5641 ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     5701 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     5761 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     5821 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     5881 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     5941 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     6001 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     6061 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     6121 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     6181 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     6241 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     6301 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     6361 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     6421 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     6481 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     6541 taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6601 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6661 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6721 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6781 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6841 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6901 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6961 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7021 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg gttcccgaag
     7081 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7141 agcttccagg gggaaacgcc tggtatcttt atagtcctgt
//
