LOCUS       sequence-381114-        7142 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 7142)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7142)
  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.3
FEATURES             Location/Qualifiers
     source          1..7142
                     /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"
     polyA_signal    4350..4574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4644..4725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4849..4930
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5079..5112)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5118..5149
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5154..5187
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5367..5385
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5408..5595,5596..5607)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5661..5680)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5672..6481
                     /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(5739..5758)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6624..7142,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7113..7132
                     /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 cggaattcct cgagaccatg agcggaggtg aggagctgtt cgccggcatc
     3661 gtgcccgtgc tgatcgagct ggacggcgac gtgcacggcc acaagttcag cgtgcgcggc
     3721 gagggcgagg gcgacgccga ctacggcaag ctggagatca agttcatctg caccaccggc
     3781 aagctgcccg tgccctggcc caccctggtg accaccctct gctacggcat ccagtgcttc
     3841 gcccgctacc ccgagcacat gaagatgaac gacttcttca agagcgccat gcccgagggc
     3901 tacatccagg agcgcaccat ccagttccag gacgacggca agtacaagac ccgcggcgag
     3961 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcaa ggacttcaag
     4021 gaggacggca acatcctggg ccacaagctg gagtacagct tcaacagcca caacgtgtac
     4081 atccgccccg acaaggccaa caacggcctg gaggctaact tcaagacccg ccacaacatc
     4141 gagggcggcg gcgtgcagct ggccgaccac taccagacca acgtgcccct gggcgacggc
     4201 cccgtgctga tccccatcaa ccactacctg agcactcaga ccaagatcag caaggaccgc
     4261 aacgaggccc gcgaccacat ggtgctcctg gagtccttca gcgcctgctg ccacacccac
     4321 ggcatggacg agctgtatag gtaatagatc tgtgccttct agttgccagc catctgttgt
     4381 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4441 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4501 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4561 ggtgggctct atggcttctg aggcggaaag aaccaaaggg cgaattaatt cattgatgag
     4621 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4681 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4741 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4801 ctctacaaat gtggtcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4861 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4921 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     4981 ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattcagatc aattaaccct
     5041 cacccgggct agcgatgcat gggagctcaa gacgtctgat aacttcgtat agcatacatt
     5101 atacgaagtt atctacctaa ctttaaataa tgccaattat ttaaagttac ttccgcgccc
     5161 ggggagccca agggcacgcc ctggcacctg acgtacgttg tctagcaagg aagtgaagag
     5221 ctatccggat atatatgtat gttatcataa ctgtactggc tatgtcaggt ggtaatgact
     5281 gtaacgttac gttactcgtt gtaggcacga cattgaatat gctcttctcc gctgtggccc
     5341 gaacgcctcc cgctagcgat gggatctaat acgactcact atagggagac caagggaaaa
     5401 tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     5461 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     5521 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     5581 aaattactca agtactttac acctctggga tccagctgca tcatgaacaa taaaactgtc
     5641 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     5701 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     5761 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     5821 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     5881 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     5941 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     6001 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     6061 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6121 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6181 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     6241 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     6301 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     6361 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     6421 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     6481 atcagaattg gttaattggt tgtaacactg gcaaaaagga tctaggtgaa gatccttttt
     6541 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6601 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6661 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6721 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6781 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6841 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6901 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6961 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7021 gaaagcgcca cggttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7081 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7141 gt
//
