LOCUS       sequence-362198-        8535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockin plasmid with homologous arm for making zMADM-GG zebrafish 
            line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8535)
  AUTHORS   Xu B, Kucenas S, Zong H
  TITLE     zMADM (zebrafish mosaic analysis with double markers) for 
            single-cell gene knockout and dual-lineage tracing.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Mar 1;119(9). pii: 2122529119. doi: 
            10.1073/pnas.2122529119.
  PUBMED    35197298
REFERENCE   2  (bases 1 to 8535)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2022-03-1"; volume: "119(9). pii: 
            2122529119. doi"; pages: " 10.1073/pnas.2122529119"
COMMENT     Sequence Label: KI-eab2-GG
FEATURES             Location/Qualifiers
     source          1..8535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    349..382
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    383..416
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    417..450
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(485..518)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    521..552
                     /label=rox
                     /bound_moiety="Dre recombinase"
     primer_bind     777..798
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      840..849
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4570..4732,4733..4744)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     5093..5114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5277..5381
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5382..5450,5451..6242)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5600..5619)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6413..7001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6902..6921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7155..7172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7289..7310
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7325..7330,7331..7348,7349..7355)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7363..7379
                     /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     7368..7390
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7387..7403
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7387..7403
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7422..7442
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7424..7442
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     7479..7495
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7479..7495
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(7529..7540,7541..7728)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    8167..8301
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8204..8223)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8258..8277
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8364..8385)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tcgccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtaccc taagggtggg
      241 aaaatagacc aataggcaga gagagtcagt gcctatcaga aacccaagag tcttctctgt
      301 ctccacatgc ccagtttcta ttggtctcct taaacctgtc ttgtaaccat aacttcgtat
      361 aatgtatgct atacgaagtt atataacttc gtataaagta tcctatacga agttatataa
      421 cttcgtataa tgtgtactat acgaagttat gaagttccta taccttttga agaataggaa
      481 cttcgaagtt cctatacttt ctagagaata ggaacttcgg taactttaaa taattggcat
      541 tatttaaagt taagatcttt gataccaacc ttcgggcatg gcggacttga agaagtcgtg
      601 ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca gggtggtcac
      661 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
      721 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
      781 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
      841 catggtggcg gatccactgt aaaagaaagg gaaactgtta gaattgcact gctgccaggg
      901 agaggcgttt cagcaagcat gtgaccatct ggagtcaact tcctgttttg ctctgcattc
      961 tacagtctga ccgctggtgg gcgctagtga cagcgcgcgc ctcagatatg ccagacaaag
     1021 gaagtccctc tgcattctct cattattacc ataaaaggca atggtttgag ccgctttgcg
     1081 gctgccggcg gtgtggcact cagccagagt cgtagggctc agtaaaaagg aacagatttc
     1141 gacattcatc acaccaggac cactccctaa attttacaca aatgaagttt cacatcctct
     1201 tccaccctcg cctgcgatca attacacaac cgcatttcaa cgggtgtctg ccgccgggtg
     1261 ttactgcgtc acagatctgc agtcagtcca cggactcctc gtgagcggag acttcacact
     1321 aaagcatttc tggacggtca ctctcagtcg gttcagttca tagtctagcc tcaaaaatca
     1381 ccgagatcct gaaaaactga cgggctctgc ttcgacagtc acagcggctg aagtgcagcc
     1441 agagaactcc gcagccgtta ttaaagtgga ccacacctct aaaattcaaa actctttcat
     1501 agcggataca atacttcact aacgttaatc ttcagcttca ttaaagacgg agggggctgc
     1561 aggctacatt taaatagaac acataacaaa aagaaaatta accaagttca aactaaatgc
     1621 caatacccgt tactaataac aagatcttcg atcttataaa gcacgaatgg gccgatgtta
     1681 gattgatggg gtttacgtta catcagatga gagctggtta gccggctagc taaagtaggt
     1741 cacaccctgc tgcagcactg cctgcacaaa gaacttccca tgccggctat ccatcaagtg
     1801 tgcaacatgc tcaccagcac cctcaaaact gtagttaaaa atcacattta tggctgttga
     1861 agagagatta gccagaacac aactttatat taagacttaa cacccatttc acaccggccc
     1921 attcaatgtt caatgtcgtg cctacgactg gtcacgttaa attaccgaca ttctcacctg
     1981 ccacatccac taacgctaca aaaaaagtta agatacagcc aatatttatt aaatgtaaca
     2041 gatattatca aaagaaccac catttataaa tcaaacctag tagataacta caaagagacc
     2101 gttatgaact gtctaaagac ttcatcaaat catatttatc catttacaca ttttacttaa
     2161 cattaataga tagaaactat aattaaacgc ttaaaaactg caaaaccaac tcaccttggg
     2221 tttggggggt tgtcgactta agagaatggg ttacaagcag atgatattcc gcactagctg
     2281 cgtgtggagg agctcaaagt gaggctgaga cgctgggcgc gctcttttat atgacggcac
     2341 atacccggct ctagccataa aaggtaaact ttcggagctc tgtgctctga ttggtccagc
     2401 gtcacacacc cggctgtcgc gtgcacattg ctacacttgc ttcttgcaac ttgagagtga
     2461 aagaagggga agcttaactg gttaaagaac aaggccttgg cgatttccta taaaactgga
     2521 atttttattt gcaaatttta acacagaatt tttcgtattt ttctgcgaca acgccaagac
     2581 tcaattcaac gtttcaaaaa gtctgtgcag agttacaagt ggcagaatgc gtcataatga
     2641 tggatgcctc catgatcctg tgttgtgttt tcaatctaac acaacagcag ctctgtaggt
     2701 catttacctc ttgtgttcac gaaataccgt aatacaaaca atgggggatc taatgagtac
     2761 ttttccatgt ataaaatgga catttttttt atccaaatta ggatgacact acttgtagta
     2821 tgttgtagga gcaactagct ggtcttagct cagtagtaat gcactttaag aaagtgcttt
     2881 aataactttg aatgaatggg acctgcattc catgccacat tgcttcacta tttttgtgtg
     2941 tacaagttac acaatgatgt ataaagtgag tgatagcagt tacatttttg caagtgggcc
     3001 tacctcaggt ttaatgttca ttagaaatgt tatatgtcaa gtcttggtct taaatagtta
     3061 aattttctgt tcataatttc atgtcgtgtc tatgcagaca taacatttct ttttaaagag
     3121 gtttataagg ataaatccta agaacataat tctcattaaa agtagaaaaa aaaacactga
     3181 aagtgttggt gatcagctac aataaataat attttgcaca catttcagat gttaccacac
     3241 tgaataacag ataaagctgt gcttgagcag cagtgatcat gataaactac atgtgacttt
     3301 ttactgtgta acttgggagt aatgcacatg gccttcagtg ggagttataa acaaaaacag
     3361 ctgattgtgt taaaagtgct tgctgatacc aaggacaatg aggtgatgtg acttttattt
     3421 tgttttcggc aaattccaag tattttgaca agacttttta caattctgag cattttgcct
     3481 ctaaaatatg gaagtttctc agagtaaacg ttgagtatag gtaatgccta ggccctgact
     3541 gtgcgcacag aaatctgtag atttccccag atctttagcc catcattgag tttatttatt
     3601 gacttgtgta tttggatttg ctctttagtg tttggactct cagcagtaaa aattaaccca
     3661 cactgaacta aactgaactg aacttaaact cttaaaaatg gactgacaca gtttcaattt
     3721 actatactaa tcttctaagt taagctgctt tgacacaatc tacattgtaa aagcactcta
     3781 gcttgcatgc ctgagaattt cagaatgtaa tgataccttt gttagataag gctgtttaca
     3841 tctgatagtg gaccttaagc cgacacttaa atgataaaaa cggcaaagaa ttgcaagttt
     3901 gatttgcatt ggaaagggtc gctggctttt gtgttacacg ccccttattt gtgcttgatt
     3961 agatgatccc cctgctcgag tttccttaca ggcctaaatt tggatttcta atatcacttc
     4021 accttcctct aaagatactg acagcctaag aaagcacttg ggtccagtta agccgcctat
     4081 tgatcagact tgaagtaaaa atctatttaa tggcctgaaa cgcgatcata cattgaaaaa
     4141 caccaacata agatccgcga aacattgcca aaagcagttt tagagagtga aaagtgagaa
     4201 tgaagatttc agctaaaggt gataaatggt gatgttaatg gtgcagaaca gaaggggctc
     4261 aagttttcaa ccccattaca taagcagtcc aaggctaact caacctacca gccatacaaa
     4321 ttgattgagt aattttgtcc acgaaaggat ctcaccttca agccaacctc cagcaaccaa
     4381 aaataaacta aaaaacctca tttatcttct aactctcact ctcacgcttc acatacgctg
     4441 attggggaga gacttaagag tgttgctcct gcagtcacgt taaagcacag ggtggtgaac
     4501 tcatctggaa atgcatttca tttaatgttt tttgagatta aaagcttaaa caagaatctc
     4561 tagttttctt tcttgctttt acttttactt ccttaatact caagtacaat tttaatggag
     4621 tactttttta cttttactca agtaagattc tagccagata cttttacttt taattgagta
     4681 aaattttccc taagtacttg tactttcact tgagtaaaat ttttgagtac tttttacacc
     4741 tctgtcaaga actcctggac aaacctctga cctgtgtgga acagagtgga tatgggtgtc
     4801 tgaacagata ttcacgtctt ttgcagatca gagggcattt ctggtgttgt gcaatgacct
     4861 gggtccaaca ggtaggcgta ctcgtatcca agagcattgt tggacaggag gtagtctcca
     4921 ttcctataaa gaggaaggaa tggcaccatg tagtagccgt cattgtggcc aattggtgca
     4981 ttggtacgtg ggtagatgga ccggggaggc tgatgagttc ttagccagcg tgggccatcg
     5041 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5101 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     5161 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5221 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     5281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5341 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5401 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5461 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5521 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5581 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5641 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5701 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5761 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5821 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5881 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5941 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6001 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6061 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6121 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6181 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6241 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6301 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatg ccttaacgtg
     6361 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6421 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6481 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6541 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6601 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6661 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6721 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6781 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6841 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6901 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6961 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7021 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7081 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7141 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     7201 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     7261 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     7321 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     7381 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     7441 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatctgctg
     7501 ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa gtacttgagt aattttactt
     7561 gattactgta cttaagtatt atttttgggg atttttactt tacttgagta caattaaaaa
     7621 tcaatacttt tacttttact taattacatt tttttagaaa aaaaagtact ttttactcct
     7681 tacaatttta tttacagtca aaaagtactt attttttgga gatcacttca ttctattttc
     7741 ccttgctatt accaaaccaa ttgaattgcg ctgatgccca gtttaattta aatgttattt
     7801 attctgccta tgaaaatcgt tttcacatta tatgaaattg gtcagacatg ttcattggtc
     7861 ctttggaagt gacgtcatgt cacatctatt accacaatgc acagcacctt gacctggaaa
     7921 ttagggaaat tataacagtc aatcagtgga agaaaatgga ggaagtatgt gattcatcag
     7981 cagctgcgag cagcacagtc caaaatcagc cacaggatca agagcacccg tggccgtatc
     8041 ttcgcagatc tgatctagag gatcataatc agccatacca catttgtaga ggttttactt
     8101 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     8161 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     8221 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     8281 gtatcttatc atgtcgatat caagtatact tacatatgtt acttgtaaag ctcgtccatg
     8341 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     8401 ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg
     8461 gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gccgccgtcc
     8521 tcgatgttgt ggcgg
//
