LOCUS       sequence-380758-        7276 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 7276)
  TITLE     Clark Lab Gene Break Homology Plasmids 2022
REFERENCE   2  (bases 1 to 7276)
  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-gcryBFP.1
FEATURES             Location/Qualifiers
     source          1..7276
                     /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             946..1620
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             946..1620
                     /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(1073..1091)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1341..1360
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1535..1558
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3495..3515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3519..4220
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4230..4454
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4524..4605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4729..4810
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4959..4992)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4998..5029
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    5034..5067
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        5247..5265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5288..5475,5476..5487)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5541..5560)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5552..6361
                     /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(5619..5638)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6504..7092
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6993..7012
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7246..7263
                     /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 cagttcagcc ggaattcacc cgggtaccgg aaggtgcctc ctccgaggac
      961 gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     1021 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1081 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc tcagttccag
     1141 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1201 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1261 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1321 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1381 accgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagat gaggctgaag
     1441 ctgaaggacg gcggccacta cgacgccgag gtcaagacca cctacatggc caagaagccc
     1501 gtgcagctgc ccggcgccta caagaccgac atcaagctgg acatcacctc ccacaacgag
     1561 gactacacca tcgtggaaca gtacgagcgc gccgagggcc gccactccac cggagcctaa
     1621 gactagtaat taagtctcag ccaccgttaa ctgaacatgt caaaacctgt ggagactgtt
     1681 gagatttgat gttctgaaaa gataaagcct ataaataaaa tgttgcccaa atttcctgcc
     1741 tgatgttttt ctttgtcttt gctacatggc tttgctgctc ggatcggctc actctgtgta
     1801 tgccacgttc actttgtact ctccttctca cggtaggttt attattttta gatgtgcagt
     1861 tagtttctgt gaaataacac accacacact gatattgtct gtgcattgac ttggtgagtg
     1921 cacattgttt ttgatcttga catatttata tttgattgat caggtgaact gtgtgaatct
     1981 aaagtgctcc atacagatgt tctgcattga aaatattctc attttattag tggaagtgag
     2041 tgtatgccac atccaatcaa tttcagcaaa caccccagta tgatttaatg caaaaaaatg
     2101 aaggtatcaa acacgcatta ctactttgca gttaaatatt taacatttat tccaacacga
     2161 aaaaaagcag taaataacac tttgacaaac acgtcaggac atcttatttt tgtcaccctc
     2221 acaggcaatt tagtataata tattatatat atatatatat catataataa tattcagtat
     2281 aatatatata tatatatcat attataatat tcagtataat ataaaacaca aacacatata
     2341 tgtataatat aatataacat ttttatttat tgagatgcct ctatggaccg tgttataaga
     2401 agtaaagatc aggagaagta aacatgaagt gtaattatga atactgatgt taaattaagc
     2461 tatgatgagt tttcactgtt aatttaccat ctcaattaaa tgttgatgcc tccatgacca
     2521 agttaagcag atgagactga ggacaactgt agaagacaag atgttcactt tgctgaatat
     2581 agctggcttg acagttatct atgactctat aaatatatat atattttttt ttttataaaa
     2641 tgatttattt ataactatat atccatttct cagacaggtg cttcatatcc ctcactcccg
     2701 tagctgtcca tgctggatct gtccccgttg tttttaaaaa gctaaataag ttattaacat
     2761 gactgcatcc agcgagccaa acctgtctgg tgtacagcta ccagagaagc ttgagatcct
     2821 agtcaccggc gttcctagtt ctttaaactg ttacaaagtg tttggaccag caactgaatt
     2881 gcactgtaaa ttaccacatc agggtctccg gggtcaacgc tgctaagcaa tggactggaa
     2941 aatttgtgac attgagtttt tggaataact gggaatgctg gataactctt tgggtaaagc
     3001 tatggaacaa aagtaccagc atgaactaag aaatgactgt cacagcacaa catcaaacac
     3061 aatgcaagac aaaggttctc tgcttttgcc ttacttgaca gaaaaagtaa gctctaggtt
     3121 catcgcatgc agacagcagc aatacatgac atatataaag gctggctctt ttctcccttc
     3181 cattgaactg aaacttccac tcagtcagac ttgcggggga tccacagcct gactaactcg
     3241 acaaaccccc ccaaacctaa ggtgagttga tctttaagct ttttacattt tcagctcgca
     3301 tatatcaatt cgcgcgcatt gtcacactag cacccactag cggtcagact gcagattgca
     3361 gcacgaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt gacttccctg
     3421 acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc ggaattcacc
     3481 tcgagaccat ggctccaaag aagaagcgta aggtaaccat gagcgagctg attaaggaga
     3541 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     3601 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     3661 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     3721 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct
     3781 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     3841 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat
     3901 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt
     3961 accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga
     4021 gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca
     4081 agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg
     4141 agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac
     4201 tggggcacaa gctcaattaa agctgagatc tgtgccttct agttgccagc catctgttgt
     4261 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4321 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4381 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4441 ggtgggctct atggcttctg aggcggaaag aaccaaaggg cgaattaatt cattgatgag
     4501 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     4561 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4621 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4681 ctctacaaat gtggtcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4741 atgctttatt tgtgaaattt gtgatgctat tactttattt gtaaccatta taagctgcaa
     4801 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     4861 ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattcagatc aattaaccct
     4921 cacccgggct agcgatgcat gggagctcaa gacgtctgat aacttcgtat agcatacatt
     4981 atacgaagtt atctacctaa ctttaaataa tgccaattat ttaaagttac ttccgcgccc
     5041 ggggagccca agggcacgcc ctggcacctg acgtacgttg tctagcaagg aagtgaagag
     5101 ctatccggat atatatgtat gttatcataa ctgtactggc tatgtcaggt ggtaatgact
     5161 gtaacgttac gttactcgtt gtaggcacga cattgaatat gctcttctcc gctgtggccc
     5221 gaacgcctcc cgctagcgat gggatctaat acgactcact atagggagac caagggaaaa
     5281 tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     5341 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     5401 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     5461 aaattactca agtactttac acctctggga tccagctgca tcatgaacaa taaaactgtc
     5521 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     5581 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     5641 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     5701 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     5761 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     5821 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     5881 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     5941 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6001 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6061 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     6121 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     6181 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     6241 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     6301 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     6361 atcagaattg gttaattggt tgtaacactg gcaaaaagga tctaggtgaa gatccttttt
     6421 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6481 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6541 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6601 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6661 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6721 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6781 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6841 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6901 gaaagcgcca cggttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6961 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7021 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7081 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7141 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7201 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     7261 gaggaagcgg aacaga
//
