LOCUS       sequence-380759-        7288 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 7288)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7288)
  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.2
FEATURES             Location/Qualifiers
     source          1..7288
                     /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             945..1619
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             945..1619
                     /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(1072..1090)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1340..1359
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1534..1557
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3494..3514
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3518..3520,3521..3523,3524..4237)
                     /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(3518..3520,3521..3523,3524..4234)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3563..3584)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3824..3843)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4171..4192
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4242..4466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4536..4617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4741..4822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4971..5004)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5010..5041
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5046..5079
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5259..5277
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5300..5487,5488..5499)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5553..5572)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5564..6373
                     /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(5631..5650)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6516..7104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7005..7024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7258..7275
                     /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 cagtcagccg gaattcaccc gggtaccgga aggtgcctcc tccgaggacg
      961 tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt
     1021 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     1081 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttccagt
     1141 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     1201 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     1261 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     1321 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     1381 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg aggctgaagc
     1441 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacatggcc aagaagcccg
     1501 tgcagctgcc cggcgcctac aagaccgaca tcaagctgga catcacctcc cacaacgagg
     1561 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggagcctaag
     1621 actagtaatt aagtctcagc caccgttaac tgaacatgtc aaaacctgtg gagactgttg
     1681 agatttgatg ttctgaaaag ataaagccta taaataaaat gttgcccaaa tttcctgcct
     1741 gatgtttttc tttgtctttg ctacatggct ttgctgctcg gatcggctca ctctgtgtat
     1801 gccacgttca ctttgtactc tccttctcac ggtaggttta ttatttttag atgtgcagtt
     1861 agtttctgtg aaataacaca ccacacactg atattgtctg tgcattgact tggtgagtgc
     1921 acattgtttt tgatcttgac atatttatat ttgattgatc aggtgaactg tgtgaatcta
     1981 aagtgctcca tacagatgtt ctgcattgaa aatattctca ttttattagt ggaagtgagt
     2041 gtatgccaca tccaatcaat ttcagcaaac accccagtat gatttaatgc aaaaaaatga
     2101 aggtatcaaa cacgcattac tactttgcag ttaaatattt aacatttatt ccaacacgaa
     2161 aaaaagcagt aaataacact ttgacaaaca cgtcaggaca tcttattttt gtcaccctca
     2221 caggcaattt agtataatat attatatata tatatatatc atataataat attcagtata
     2281 atatatatat atatatcata ttataatatt cagtataata taaaacacaa acacatatat
     2341 gtataatata atataacatt tttatttatt gagatgcctc tatggaccgt gttataagaa
     2401 gtaaagatca ggagaagtaa acatgaagtg taattatgaa tactgatgtt aaattaagct
     2461 atgatgagtt ttcactgtta atttaccatc tcaattaaat gttgatgcct ccatgaccaa
     2521 gttaagcaga tgagactgag gacaactgta gaagacaaga tgttcacttt gctgaatata
     2581 gctggcttga cagttatcta tgactctata aatatatata tatttttttt tttataaaat
     2641 gatttattta taactatata tccatttctc agacaggtgc ttcatatccc tcactcccgt
     2701 agctgtccat gctggatctg tccccgttgt ttttaaaaag ctaaataagt tattaacatg
     2761 actgcatcca gcgagccaaa cctgtctggt gtacagctac cagagaagct tgagatccta
     2821 gtcaccggcg ttcctagttc tttaaactgt tacaaagtgt ttggaccagc aactgaattg
     2881 cactgtaaat taccacatca gggtctccgg ggtcaacgct gctaagcaat ggactggaaa
     2941 atttgtgaca ttgagttttt ggaataactg ggaatgctgg ataactcttt gggtaaagct
     3001 atggaacaaa agtaccagca tgaactaaga aatgactgtc acagcacaac atcaaacaca
     3061 atgcaagaca aaggttctct gcttttgcct tacttgacag aaaaagtaag ctctaggttc
     3121 atcgcatgca gacagcagca atacatgaca tatataaagg ctggctcttt tctcccttcc
     3181 attgaactga aacttccact cagtcagact tgcgggggat ccacagcctg actaactcga
     3241 caaacccccc caaacctaag gtgagttgat ctttaagctt tttacatttt cagctcgcat
     3301 atatcaattc gcgcgcattg tcacactagc acccactagc ggtcagactg cagattgcag
     3361 cacgaaacag gaagctgact ccacatggtc acatgctcac tgaagtgttg acttccctga
     3421 cagctgtgca ctttctaaac cggttttctc attcatttac agttcagccg gaattcacct
     3481 cgagaccatg gctccaaaga agaagcgtaa ggtaaccatg gtgagcaagg gcgaggagct
     3541 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3601 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3661 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     3721 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3781 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3841 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3901 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3961 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     4021 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     4081 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     4141 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     4201 cgggatcact ctcggcatgg acgagctgta caagtaaaga tctgtgcctt ctagttgcca
     4261 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4321 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4381 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     4441 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccaaag ggcgaattaa
     4501 ttcattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     4561 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     4621 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     4681 agcaagtaaa acctctacaa atgtggtcat tgatgagttt ggacaaacca caactagaat
     4741 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4801 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4861 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattcaga
     4921 tcaattaacc ctcacccggg ctagcgatgc atgggagctc aagacgtctg ataacttcgt
     4981 atagcataca ttatacgaag ttatctacct aactttaaat aatgccaatt atttaaagtt
     5041 acttccgcgc ccggggagcc caagggcacg ccctggcacc tgacgtacgt tgtctagcaa
     5101 ggaagtgaag agctatccgg atatatatgt atgttatcat aactgtactg gctatgtcag
     5161 gtggtaatga ctgtaacgtt acgttactcg ttgtaggcac gacattgaat atgctcttct
     5221 ccgctgtggc ccgaacgcct cccgctagcg atgggatcta atacgactca ctatagggag
     5281 accaagggaa aatagaatga agtgatctcc aaaaaataag tactttttga ctgtaaataa
     5341 aattgtaagg agtaaaaagt actttttttt ctaaaaaaat gtaattaagt aaaagtaaaa
     5401 gtattgattt ttaattgtac tcaagtaaag taaaaatccc caaaaataat acttaagtac
     5461 agtaatcaag taaaattact caagtacttt acacctctgg gatccagctg catcatgaac
     5521 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     5581 ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     5641 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga
     5701 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     5761 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     5821 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca
     5881 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     5941 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     6001 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     6061 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     6121 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     6181 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     6241 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     6301 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     6361 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcaaaaag gatctaggtg
     6421 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6481 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6601 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6661 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6721 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6781 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6841 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6901 cgtgagctat gagaaagcgc cacggttccc gaagggagaa aggcggacag gtatccggta
     6961 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7021 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7081 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7141 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7201 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7261 gagtcagtga gcgaggaagc ggaacaga
//
