LOCUS       sequence-381117-        7163 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 7163)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7163)
  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.3
FEATURES             Location/Qualifiers
     source          1..7163
                     /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             1198..1872
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1198..1872
                     /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(1325..1343)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1593..1612
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1787..1810
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3636..3656
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3660..4361
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4371..4595
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4665..4746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4870..4951
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5100..5133)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5139..5170
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5175..5208
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5388..5406
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5429..5616,5617..5628)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5682..5701)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5693..6502
                     /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(5760..5779)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6645..7163,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7134..7153
                     /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 tttacagcag ccggaattca cccgggtacc ggaaggtgcc
     1201 tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg
     1261 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1321 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1381 cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1441 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1501 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1561 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1621 tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1681 atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacatg
     1741 gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct ggacatcacc
     1801 tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc
     1861 accggagcct aagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct
     1921 gtggagactg ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc
     1981 aaatttcctg cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc
     2041 tcactctgtg tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt
     2101 tagatgtgca gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg
     2161 acttggtgag tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa
     2221 ctgtgtgaat ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt
     2281 agtggaagtg agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa
     2341 tgcaaaaaaa tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt
     2401 attccaacac gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt
     2461 tttgtcaccc tcacaggcaa tttagtataa tatattatat atatatatat atcatataat
     2521 aatattcagt ataatatata tatatatatc atattataat attcagtata atataaaaca
     2581 caaacacata tatgtataat ataatataac atttttattt attgagatgc ctctatggac
     2641 cgtgttataa gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat
     2701 gttaaattaa gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg
     2761 cctccatgac caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac
     2821 tttgctgaat atagctggct tgacagttat ctatgactct ataaatatat atatattttt
     2881 ttttttataa aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat
     2941 ccctcactcc cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata
     3001 agttattaac atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa
     3061 gcttgagatc ctagtcaccg gcggcattca tccatccttt tcatccctca aatctctcat
     3121 tcacgtcccc ctccccatct gcacacttta tctcattttc caccctgctg gaatctgagc
     3181 acttgtgcag ttatcagggc tcctgtattt aggaggctct gggtgtccat gtaggggacg
     3241 aacagaaaca ctgcagacct ttatagaaga acaaatgata agagtcctca tacataaaga
     3301 ctccattaga aacgtcagtg acccaggagc ccagaccaac agcaaagcag acagtgaagg
     3361 atccacagcc tgactaactc gacaaacccc cccaaaccta aggtgagttg atctttaagc
     3421 tttttacatt ttcagctcgc atatatcaat tcgcgcgcat tgtcacacta gcacccacta
     3481 gcggtcagac tgcagattgc agcacgaaac aggaagctga ctccacatgg tcacatgctc
     3541 actgaagtgt tgacttccct gacagctgtg cactttctaa accggttttc tcattcattt
     3601 acagttcagc cggaattcac ctcgagacca tggctccaaa gaagaagcgt aaggtaacca
     3661 tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca
     3721 accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     3781 tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta
     3841 gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca
     3901 agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     3961 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     4021 tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     4081 aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg
     4141 ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca tatagatcca
     4201 agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa
     4261 gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat
     4321 actgcgacct ccctagcaaa ctggggcaca agctcaatta aagctgagat ctgtgccttc
     4381 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4441 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4501 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4561 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccaaagg
     4621 gcgaattaat tcattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     4681 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4741 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     4801 gttttttaaa gcaagtaaaa cctctacaaa tgtggtcatt gatgagtttg gacaaaccac
     4861 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4921 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4981 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     5041 tcattcagat caattaaccc tcacccgggc tagcgatgca tgggagctca agacgtctga
     5101 taacttcgta tagcatacat tatacgaagt tatctaccta actttaaata atgccaatta
     5161 tttaaagtta cttccgcgcc cggggagccc aagggcacgc cctggcacct gacgtacgtt
     5221 gtctagcaag gaagtgaaga gctatccgga tatatatgta tgttatcata actgtactgg
     5281 ctatgtcagg tggtaatgac tgtaacgtta cgttactcgt tgtaggcacg acattgaata
     5341 tgctcttctc cgctgtggcc cgaacgcctc ccgctagcga tgggatctaa tacgactcac
     5401 tatagggaga ccaagggaaa atagaatgaa gtgatctcca aaaaataagt actttttgac
     5461 tgtaaataaa attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta
     5521 aaagtaaaag tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata
     5581 cttaagtaca gtaatcaagt aaaattactc aagtacttta cacctctggg atccagctgc
     5641 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     5701 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     5761 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     5821 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     5881 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     5941 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     6001 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     6061 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     6121 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     6181 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     6241 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     6301 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     6361 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     6421 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     6481 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcaaaaagg
     6541 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6601 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6661 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6721 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6781 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6841 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6901 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6961 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7021 tacctacagc gtgagctatg agaaagcgcc acggttcccg aagggagaaa ggcggacagg
     7081 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7141 gcctggtatc tttatagtcc tgt
//
