LOCUS       sequence-381137-        7164 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 7164)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7164)
  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-cmlc2BFP.2
FEATURES             Location/Qualifiers
     source          1..7164
                     /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..1873
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1199..1873
                     /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(1326..1344)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1594..1613
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1788..1811
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3637..3657
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3661..4362
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4372..4596
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4666..4747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4871..4952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5101..5134)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5140..5171
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5176..5209
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5389..5407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5430..5617,5618..5629)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5683..5702)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5694..6503
                     /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(5761..5780)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6646..7164,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7135..7154
                     /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 ctcctccgag gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt
     1261 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     1321 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc
     1381 ccctcagttc cagtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     1441 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     1501 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     1561 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     1621 ctgggaggcc tccaccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     1681 gatgaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga ccacctacat
     1741 ggccaagaag cccgtgcagc tgcccggcgc ctacaagacc gacatcaagc tggacatcac
     1801 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg gccgccactc
     1861 caccggagcc taagactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc
     1921 tgtggagact gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc
     1981 caaatttcct gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg
     2041 ctcactctgt gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt
     2101 ttagatgtgc agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt
     2161 gacttggtga gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga
     2221 actgtgtgaa tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat
     2281 tagtggaagt gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta
     2341 atgcaaaaaa atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt
     2401 tattccaaca cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat
     2461 ttttgtcacc ctcacaggca atttagtata atatattata tatatatata tatcatataa
     2521 taatattcag tataatatat atatatatat catattataa tattcagtat aatataaaac
     2581 acaaacacat atatgtataa tataatataa catttttatt tattgagatg cctctatgga
     2641 ccgtgttata agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga
     2701 tgttaaatta agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat
     2761 gcctccatga ccaagttaag cagatgagac tgaggacaac tgtagaagac aagatgttca
     2821 ctttgctgaa tatagctggc ttgacagtta tctatgactc tataaatata tatatatttt
     2881 tttttttata aaatgattta tttataacta tatatccatt tctcagacag gtgcttcata
     2941 tccctcactc ccgtagctgt ccatgctgga tctgtccccg ttgtttttaa aaagctaaat
     3001 aagttattaa catgactgca tccagcgagc caaacctgtc tggtgtacag ctaccagaga
     3061 agcttgagat cctagtcacc ggcggcattc atccatcctt ttcatccctc aaatctctca
     3121 ttcacgtccc cctccccatc tgcacacttt atctcatttt ccaccctgct ggaatctgag
     3181 cacttgtgca gttatcaggg ctcctgtatt taggaggctc tgggtgtcca tgtaggggac
     3241 gaacagaaac actgcagacc tttatagaag aacaaatgat aagagtcctc atacataaag
     3301 actccattag aaacgtcagt gacccaggag cccagaccaa cagcaaagca gacagtgaag
     3361 gatccacagc ctgactaact cgacaaaccc ccccaaacct aaggtgagtt gatctttaag
     3421 ctttttacat tttcagctcg catatatcaa ttcgcgcgca ttgtcacact agcacccact
     3481 agcggtcaga ctgcagattg cagcacgaaa caggaagctg actccacatg gtcacatgct
     3541 cactgaagtg ttgacttccc tgacagctgt gcactttcta aaccggtttt ctcattcatt
     3601 tacagttcag ccggaattca cctcgagacc atggctccaa agaagaagcg taaggtaacc
     3661 atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac
     3721 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     3781 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact
     3841 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc
     3901 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     3961 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     4021 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg
     4081 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg
     4141 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc
     4201 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa
     4261 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga
     4321 tactgcgacc tccctagcaa actggggcac aagctcaatt aaagctgaga tctgtgcctt
     4381 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4441 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4501 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4561 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccaaag
     4621 ggcgaattaa ttcattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     4681 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     4741 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     4801 ggttttttaa agcaagtaaa acctctacaa atgtggtcat tgatgagttt ggacaaacca
     4861 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     4921 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     4981 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     5041 gtcattcaga tcaattaacc ctcacccggg ctagcgatgc atgggagctc aagacgtctg
     5101 ataacttcgt atagcataca ttatacgaag ttatctacct aactttaaat aatgccaatt
     5161 atttaaagtt acttccgcgc ccggggagcc caagggcacg ccctggcacc tgacgtacgt
     5221 tgtctagcaa ggaagtgaag agctatccgg atatatatgt atgttatcat aactgtactg
     5281 gctatgtcag gtggtaatga ctgtaacgtt acgttactcg ttgtaggcac gacattgaat
     5341 atgctcttct ccgctgtggc ccgaacgcct cccgctagcg atgggatcta atacgactca
     5401 ctatagggag accaagggaa aatagaatga agtgatctcc aaaaaataag tactttttga
     5461 ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat gtaattaagt
     5521 aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc caaaaataat
     5581 acttaagtac agtaatcaag taaaattact caagtacttt acacctctgg gatccagctg
     5641 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     5701 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
     5761 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
     5821 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     5881 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     5941 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
     6001 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     6061 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     6121 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     6181 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     6241 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     6301 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     6361 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     6421 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     6481 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac tggcaaaaag
     6541 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6661 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6721 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6781 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6841 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6901 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6961 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7021 atacctacag cgtgagctat gagaaagcgc cacggttccc gaagggagaa aggcggacag
     7081 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7141 cgcctggtat ctttatagtc ctgt
//
