LOCUS       sequence-381130-        7289 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 7289)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7289)
  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.1
FEATURES             Location/Qualifiers
     source          1..7289
                     /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..1620
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             946..1620
                     /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(1073..1091)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1341..1360
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1535..1558
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3495..3515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3519..3521,3522..3524,3525..4238)
                     /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(3519..3521,3522..3524,3525..4235)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3564..3585)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3825..3844)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4172..4193
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4243..4467
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4537..4618
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4742..4823
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4972..5005)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5011..5042
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5047..5080
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5260..5278
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5301..5488,5489..5500)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5554..5573)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5565..6374
                     /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(5632..5651)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6517..7105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7006..7025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7259..7276
                     /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 aaggtgcctc ctccgaggac
      961 gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     1021 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1081 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc tcagttccag
     1141 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1201 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1261 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1321 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1381 accgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagat gaggctgaag
     1441 ctgaaggacg gcggccacta cgacgccgag gtcaagacca cctacatggc caagaagccc
     1501 gtgcagctgc ccggcgccta caagaccgac atcaagctgg acatcacctc ccacaacgag
     1561 gactacacca tcgtggaaca gtacgagcgc gccgagggcc gccactccac cggagcctaa
     1621 gactagtaat taagtctcag ccaccgttaa ctgaacatgt caaaacctgt ggagactgtt
     1681 gagatttgat gttctgaaaa gataaagcct ataaataaaa tgttgcccaa atttcctgcc
     1741 tgatgttttt ctttgtcttt gctacatggc tttgctgctc ggatcggctc actctgtgta
     1801 tgccacgttc actttgtact ctccttctca cggtaggttt attattttta gatgtgcagt
     1861 tagtttctgt gaaataacac accacacact gatattgtct gtgcattgac ttggtgagtg
     1921 cacattgttt ttgatcttga catatttata tttgattgat caggtgaact gtgtgaatct
     1981 aaagtgctcc atacagatgt tctgcattga aaatattctc attttattag tggaagtgag
     2041 tgtatgccac atccaatcaa tttcagcaaa caccccagta tgatttaatg caaaaaaatg
     2101 aaggtatcaa acacgcatta ctactttgca gttaaatatt taacatttat tccaacacga
     2161 aaaaaagcag taaataacac tttgacaaac acgtcaggac atcttatttt tgtcaccctc
     2221 acaggcaatt tagtataata tattatatat atatatatat catataataa tattcagtat
     2281 aatatatata tatatatcat attataatat tcagtataat ataaaacaca aacacatata
     2341 tgtataatat aatataacat ttttatttat tgagatgcct ctatggaccg tgttataaga
     2401 agtaaagatc aggagaagta aacatgaagt gtaattatga atactgatgt taaattaagc
     2461 tatgatgagt tttcactgtt aatttaccat ctcaattaaa tgttgatgcc tccatgacca
     2521 agttaagcag atgagactga ggacaactgt agaagacaag atgttcactt tgctgaatat
     2581 agctggcttg acagttatct atgactctat aaatatatat atattttttt ttttataaaa
     2641 tgatttattt ataactatat atccatttct cagacaggtg cttcatatcc ctcactcccg
     2701 tagctgtcca tgctggatct gtccccgttg tttttaaaaa gctaaataag ttattaacat
     2761 gactgcatcc agcgagccaa acctgtctgg tgtacagcta ccagagaagc ttgagatcct
     2821 agtcaccggc gttcctagtt ctttaaactg ttacaaagtg tttggaccag caactgaatt
     2881 gcactgtaaa ttaccacatc agggtctccg gggtcaacgc tgctaagcaa tggactggaa
     2941 aatttgtgac attgagtttt tggaataact gggaatgctg gataactctt tgggtaaagc
     3001 tatggaacaa aagtaccagc atgaactaag aaatgactgt cacagcacaa catcaaacac
     3061 aatgcaagac aaaggttctc tgcttttgcc ttacttgaca gaaaaagtaa gctctaggtt
     3121 catcgcatgc agacagcagc aatacatgac atatataaag gctggctctt ttctcccttc
     3181 cattgaactg aaacttccac tcagtcagac ttgcggggga tccacagcct gactaactcg
     3241 acaaaccccc ccaaacctaa ggtgagttga tctttaagct ttttacattt tcagctcgca
     3301 tatatcaatt cgcgcgcatt gtcacactag cacccactag cggtcagact gcagattgca
     3361 gcacgaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt gacttccctg
     3421 acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc ggaattcacc
     3481 tcgagaccat ggctccaaag aagaagcgta aggtaaccat ggtgagcaag ggcgaggagc
     3541 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3601 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     3661 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     3721 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3781 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     3841 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3901 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     3961 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     4021 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     4081 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     4141 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     4201 ccgggatcac tctcggcatg gacgagctgt acaagtaaag atctgtgcct tctagttgcc
     4261 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4321 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4381 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4441 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccaaa gggcgaatta
     4501 attcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4561 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4621 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4681 aagcaagtaa aacctctaca aatgtggtca ttgatgagtt tggacaaacc acaactagaa
     4741 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     4801 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     4861 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattcag
     4921 atcaattaac cctcacccgg gctagcgatg catgggagct caagacgtct gataacttcg
     4981 tatagcatac attatacgaa gttatctacc taactttaaa taatgccaat tatttaaagt
     5041 tacttccgcg cccggggagc ccaagggcac gccctggcac ctgacgtacg ttgtctagca
     5101 aggaagtgaa gagctatccg gatatatatg tatgttatca taactgtact ggctatgtca
     5161 ggtggtaatg actgtaacgt tacgttactc gttgtaggca cgacattgaa tatgctcttc
     5221 tccgctgtgg cccgaacgcc tcccgctagc gatgggatct aatacgactc actataggga
     5281 gaccaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg actgtaaata
     5341 aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag taaaagtaaa
     5401 agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa tacttaagta
     5461 cagtaatcaa gtaaaattac tcaagtactt tacacctctg ggatccagct gcatcatgaa
     5521 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     5581 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     5641 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
     5701 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     5761 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     5821 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc
     5881 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     5941 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     6001 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     6061 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     6121 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     6181 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     6241 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     6301 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     6361 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcaaaaa ggatctaggt
     6421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6661 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6901 gcgtgagcta tgagaaagcg ccacggttcc cgaagggaga aaggcggaca ggtatccggt
     6961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7141 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7201 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7261 cgagtcagtg agcgaggaag cggaacaga
//
