LOCUS       sequence-381139-        7326 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 7326)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7326)
  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.1
FEATURES             Location/Qualifiers
     source          1..7326
                     /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             join(3556..3558,3559..3561,3562..4275)
                     /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(3556..3558,3559..3561,3562..4272)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3601..3622)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3862..3881)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4209..4230
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4280..4504
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4574..4655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4779..4860
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5009..5042)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5048..5079
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5084..5117
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5297..5315
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5338..5525,5526..5537)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5591..5610)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5602..6411
                     /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(5669..5688)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6554..7142
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7043..7062
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7296..7313
                     /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 taaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3601 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3661 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3721 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     3781 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3841 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3901 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3961 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4021 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4081 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4141 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4201 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     4261 gagctgtaca agtaaagatc tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4321 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4381 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4441 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     4501 atggcttctg aggcggaaag aaccaaaggg cgaattaatt cattgatgag tttggacaaa
     4561 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4621 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4681 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     4741 gtggtcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     4801 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     4861 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     4921 taaagcaagt aaaacctcta caaatgtggt cattcagatc aattaaccct cacccgggct
     4981 agcgatgcat gggagctcaa gacgtctgat aacttcgtat agcatacatt atacgaagtt
     5041 atctacctaa ctttaaataa tgccaattat ttaaagttac ttccgcgccc ggggagccca
     5101 agggcacgcc ctggcacctg acgtacgttg tctagcaagg aagtgaagag ctatccggat
     5161 atatatgtat gttatcataa ctgtactggc tatgtcaggt ggtaatgact gtaacgttac
     5221 gttactcgtt gtaggcacga cattgaatat gctcttctcc gctgtggccc gaacgcctcc
     5281 cgctagcgat gggatctaat acgactcact atagggagac caagggaaaa tagaatgaag
     5341 tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag taaaaagtac
     5401 ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt aattgtactc
     5461 aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta aaattactca
     5521 agtactttac acctctggga tccagctgca tcatgaacaa taaaactgtc tgcttacata
     5581 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     5641 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     5701 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     5761 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     5821 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     5881 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     5941 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     6001 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     6061 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     6121 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     6181 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     6241 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     6301 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     6361 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     6421 gttaattggt tgtaacactg gcaaaaagga tctaggtgaa gatccttttt gataatctca
     6481 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6541 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6601 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6661 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6721 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6781 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6841 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6901 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6961 cggttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7021 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7081 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7141 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7201 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7261 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7321 aacaga
//
