LOCUS       sequence-381042-        7144 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 7144)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7144)
  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-cmlc2GFP.1
FEATURES             Location/Qualifiers
     source          1..7144
                     /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             1200..1874
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1200..1874
                     /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(1327..1345)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1595..1614
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1789..1812
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4352..4576
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4646..4727
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4851..4932
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5081..5114)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5120..5151
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5156..5189
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5369..5387
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5410..5597,5598..5609)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5663..5682)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5674..6483
                     /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(5741..5760)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6626..7144,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7115..7134
                     /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 tttacagttc agccggaatt cacccgggta ccggaaggtg
     1201 cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg
     1261 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     1321 agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
     1381 cccctcagtt ccagtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     1441 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     1501 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     1561 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     1621 gctgggaggc ctccaccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     1681 agatgaggct gaagctgaag gacggcggcc actacgacgc cgaggtcaag accacctaca
     1741 tggccaagaa gcccgtgcag ctgcccggcg cctacaagac cgacatcaag ctggacatca
     1801 cctcccacaa cgaggactac accatcgtgg aacagtacga gcgcgccgag ggccgccact
     1861 ccaccggagc ctaagactag taattaagtc tcagccaccg ttaactgaac atgtcaaaac
     1921 ctgtggagac tgttgagatt tgatgttctg aaaagataaa gcctataaat aaaatgttgc
     1981 ccaaatttcc tgcctgatgt ttttctttgt ctttgctaca tggctttgct gctcggatcg
     2041 gctcactctg tgtatgccac gttcactttg tactctcctt ctcacggtag gtttattatt
     2101 tttagatgtg cagttagttt ctgtgaaata acacaccaca cactgatatt gtctgtgcat
     2161 tgacttggtg agtgcacatt gtttttgatc ttgacatatt tatatttgat tgatcaggtg
     2221 aactgtgtga atctaaagtg ctccatacag atgttctgca ttgaaaatat tctcatttta
     2281 ttagtggaag tgagtgtatg ccacatccaa tcaatttcag caaacacccc agtatgattt
     2341 aatgcaaaaa aatgaaggta tcaaacacgc attactactt tgcagttaaa tatttaacat
     2401 ttattccaac acgaaaaaaa gcagtaaata acactttgac aaacacgtca ggacatctta
     2461 tttttgtcac cctcacaggc aatttagtat aatatattat atatatatat atatcatata
     2521 ataatattca gtataatata tatatatata tcatattata atattcagta taatataaaa
     2581 cacaaacaca tatatgtata atataatata acatttttat ttattgagat gcctctatgg
     2641 accgtgttat aagaagtaaa gatcaggaga agtaaacatg aagtgtaatt atgaatactg
     2701 atgttaaatt aagctatgat gagttttcac tgttaattta ccatctcaat taaatgttga
     2761 tgcctccatg accaagttaa gcagatgaga ctgaggacaa ctgtagaaga caagatgttc
     2821 actttgctga atatagctgg cttgacagtt atctatgact ctataaatat atatatattt
     2881 ttttttttat aaaatgattt atttataact atatatccat ttctcagaca ggtgcttcat
     2941 atccctcact cccgtagctg tccatgctgg atctgtcccc gttgttttta aaaagctaaa
     3001 taagttatta acatgactgc atccagcgag ccaaacctgt ctggtgtaca gctaccagag
     3061 aagcttgaga tcctagtcac cggcggcatt catccatcct tttcatccct caaatctctc
     3121 attcacgtcc ccctccccat ctgcacactt tatctcattt tccaccctgc tggaatctga
     3181 gcacttgtgc agttatcagg gctcctgtat ttaggaggct ctgggtgtcc atgtagggga
     3241 cgaacagaaa cactgcagac ctttatagaa gaacaaatga taagagtcct catacataaa
     3301 gactccatta gaaacgtcag tgacccagga gcccagacca acagcaaagc agacagtgaa
     3361 ggatccacag cctgactaac tcgacaaacc cccccaaacc taaggtgagt tgatctttaa
     3421 gctttttaca ttttcagctc gcatatatca attcgcgcgc attgtcacac tagcacccac
     3481 tagcggtcag actgcagatt gcagcacgaa acaggaagct gactccacat ggtcacatgc
     3541 tcactgaagt gttgacttcc ctgacagctg tgcactttct aaaccggttt tctcattcat
     3601 ttacagttca gccggaattc ctcgagacca tgagcggagg tgaggagctg ttcgccggca
     3661 tcgtgcccgt gctgatcgag ctggacggcg acgtgcacgg ccacaagttc agcgtgcgcg
     3721 gcgagggcga gggcgacgcc gactacggca agctggagat caagttcatc tgcaccaccg
     3781 gcaagctgcc cgtgccctgg cccaccctgg tgaccaccct ctgctacggc atccagtgct
     3841 tcgcccgcta ccccgagcac atgaagatga acgacttctt caagagcgcc atgcccgagg
     3901 gctacatcca ggagcgcacc atccagttcc aggacgacgg caagtacaag acccgcggcg
     3961 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc aaggacttca
     4021 aggaggacgg caacatcctg ggccacaagc tggagtacag cttcaacagc cacaacgtgt
     4081 acatccgccc cgacaaggcc aacaacggcc tggaggctaa cttcaagacc cgccacaaca
     4141 tcgagggcgg cggcgtgcag ctggccgacc actaccagac caacgtgccc ctgggcgacg
     4201 gccccgtgct gatccccatc aaccactacc tgagcactca gaccaagatc agcaaggacc
     4261 gcaacgaggc ccgcgaccac atggtgctcc tggagtcctt cagcgcctgc tgccacaccc
     4321 acggcatgga cgagctgtat aggtaataga tctgtgcctt ctagttgcca gccatctgtt
     4381 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4441 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4501 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4561 gcggtgggct ctatggcttc tgaggcggaa agaaccaaag ggcgaattaa ttcattgatg
     4621 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4681 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4741 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     4801 acctctacaa atgtggtcat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     4861 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     4921 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     4981 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattcaga tcaattaacc
     5041 ctcacccggg ctagcgatgc atgggagctc aagacgtctg ataacttcgt atagcataca
     5101 ttatacgaag ttatctacct aactttaaat aatgccaatt atttaaagtt acttccgcgc
     5161 ccggggagcc caagggcacg ccctggcacc tgacgtacgt tgtctagcaa ggaagtgaag
     5221 agctatccgg atatatatgt atgttatcat aactgtactg gctatgtcag gtggtaatga
     5281 ctgtaacgtt acgttactcg ttgtaggcac gacattgaat atgctcttct ccgctgtggc
     5341 ccgaacgcct cccgctagcg atgggatcta atacgactca ctatagggag accaagggaa
     5401 aatagaatga agtgatctcc aaaaaataag tactttttga ctgtaaataa aattgtaagg
     5461 agtaaaaagt actttttttt ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt
     5521 ttaattgtac tcaagtaaag taaaaatccc caaaaataat acttaagtac agtaatcaag
     5581 taaaattact caagtacttt acacctctgg gatccagctg catcatgaac aataaaactg
     5641 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg
     5701 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     5761 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag
     5821 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     5881 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     5941 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa
     6001 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     6061 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     6121 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     6181 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     6241 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     6301 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     6361 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     6421 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     6481 taatcagaat tggttaattg gttgtaacac tggcaaaaag gatctaggtg aagatccttt
     6541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7021 gagaaagcgc cacggttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7081 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7141 ctgt
//
