LOCUS       sequence-381121-        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-R-gcryGFP.3
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             944..1618
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             944..1618
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(1071..1089)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1339..1358
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1533..1556
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3493..3513
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3517..3519,3520..3522,3523..4236)
                     /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(3517..3519,3520..3522,3523..4233)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3562..3583)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3823..3842)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4170..4191
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     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 cagcagccgg aattcacccg ggtaccggaa ggtgcctcct ccgaggacgt
      961 catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt
     1021 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     1081 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc agttccagta
     1141 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     1201 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     1261 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     1321 caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     1381 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga ggctgaagct
     1441 gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca agaagcccgt
     1501 gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc acaacgagga
     1561 ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg gagcctaaga
     1621 ctagtaatta agtctcagcc accgttaact gaacatgtca aaacctgtgg agactgttga
     1681 gatttgatgt tctgaaaaga taaagcctat aaataaaatg ttgcccaaat ttcctgcctg
     1741 atgtttttct ttgtctttgc tacatggctt tgctgctcgg atcggctcac tctgtgtatg
     1801 ccacgttcac tttgtactct ccttctcacg gtaggtttat tatttttaga tgtgcagtta
     1861 gtttctgtga aataacacac cacacactga tattgtctgt gcattgactt ggtgagtgca
     1921 cattgttttt gatcttgaca tatttatatt tgattgatca ggtgaactgt gtgaatctaa
     1981 agtgctccat acagatgttc tgcattgaaa atattctcat tttattagtg gaagtgagtg
     2041 tatgccacat ccaatcaatt tcagcaaaca ccccagtatg atttaatgca aaaaaatgaa
     2101 ggtatcaaac acgcattact actttgcagt taaatattta acatttattc caacacgaaa
     2161 aaaagcagta aataacactt tgacaaacac gtcaggacat cttatttttg tcaccctcac
     2221 aggcaattta gtataatata ttatatatat atatatatca tataataata ttcagtataa
     2281 tatatatata tatatcatat tataatattc agtataatat aaaacacaaa cacatatatg
     2341 tataatataa tataacattt ttatttattg agatgcctct atggaccgtg ttataagaag
     2401 taaagatcag gagaagtaaa catgaagtgt aattatgaat actgatgtta aattaagcta
     2461 tgatgagttt tcactgttaa tttaccatct caattaaatg ttgatgcctc catgaccaag
     2521 ttaagcagat gagactgagg acaactgtag aagacaagat gttcactttg ctgaatatag
     2581 ctggcttgac agttatctat gactctataa atatatatat attttttttt ttataaaatg
     2641 atttatttat aactatatat ccatttctca gacaggtgct tcatatccct cactcccgta
     2701 gctgtccatg ctggatctgt ccccgttgtt tttaaaaagc taaataagtt attaacatga
     2761 ctgcatccag cgagccaaac ctgtctggtg tacagctacc agagaagctt gagatcctag
     2821 tcaccggcgt tcctagttct ttaaactgtt acaaagtgtt tggaccagca actgaattgc
     2881 actgtaaatt accacatcag ggtctccggg gtcaacgctg ctaagcaatg gactggaaaa
     2941 tttgtgacat tgagtttttg gaataactgg gaatgctgga taactctttg ggtaaagcta
     3001 tggaacaaaa gtaccagcat gaactaagaa atgactgtca cagcacaaca tcaaacacaa
     3061 tgcaagacaa aggttctctg cttttgcctt acttgacaga aaaagtaagc tctaggttca
     3121 tcgcatgcag acagcagcaa tacatgacat atataaaggc tggctctttt ctcccttcca
     3181 ttgaactgaa acttccactc agtcagactt gcgggggatc cacagcctga ctaactcgac
     3241 aaaccccccc aaacctaagg tgagttgatc tttaagcttt ttacattttc agctcgcata
     3301 tatcaattcg cgcgcattgt cacactagca cccactagcg gtcagactgc agattgcagc
     3361 acgaaacagg aagctgactc cacatggtca catgctcact gaagtgttga cttccctgac
     3421 agctgtgcac tttctaaacc ggttttctca ttcatttaca gttcagccgg aattcacctc
     3481 gagaccatgg ctccaaagaa gaagcgtaag gtaaccatgg tgagcaaggg cgaggagctg
     3541 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3601 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3661 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3721 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3781 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3841 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3901 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3961 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     4021 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     4081 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     4141 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     4201 gggatcactc tcggcatgga cgagctgtac aagtaaagat 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
//
