LOCUS       sequence-352447-        7856 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 7856)
  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 7856)
  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..7856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      4420..4429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4471..4492)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    4717..4748
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    4751..4784
                     /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    complement(4819..4852)
                     /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(4853..4886)
                     /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    complement(4887..4920)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5072..5091)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5419..5440
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5503..5637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5527..5546)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5581..5600
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(6076..6263,6264..6275)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6309..6325)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6309..6325)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6362..6382)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6362..6380)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6401..6417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6401..6417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6414..6436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6425..6441
                     /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)."
     promoter        complement(join(6449..6455,6456..6473,6474..6479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6494..6515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6632..6649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6803..7391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6883..6902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 aggctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcatttc cagatgagtt caccaccctg tgctttaacg
      781 tgactgcagg agcaacactc ttaagtctct ccccaatcag cgtatgtgaa gcgtgagagt
      841 gagagttaga agataaatga ggttttttag tttatttttg gttgctggag gttggcttga
      901 aggtgagatc ctttcgtaga caaaattact caatcaattt gtatggctgg taggttgagt
      961 tagccttgga ctgcttatgt aatggggttg aaaacttgag ccccttctgt tctgcaccat
     1021 taacatcacc atttatcacc tttagctgaa atcttcattc tcacttttca ctctctaaaa
     1081 ctgcttttgg caatgtttcg cggatcttat gttggtgttt ttcaatgtat gatcgcgttt
     1141 caggccatta aatggatttt tacttcaagt ctgatcaata ggcggcttaa ctggacccaa
     1201 gtgctttctt aggctgtcag tatctttaga ggaaggtgaa gtgatattag aaatccaaat
     1261 ttaggcctgt aaggaaactc gagcaggggg atcatctaat caagcacaaa taaggggcgt
     1321 gtaacacaaa agccagcgac cctttccaat gcaaatcaaa cttgcaattc tttgccgttt
     1381 ttatcattta agtgtcggct taaggtccac tatcagatgt aaacagcctt atctaacaaa
     1441 ggtatcatta cattctgaaa ttctcaggca tgcaagctag agtgctttta caatgtagat
     1501 tgtgtcaaag cagcttaact tagaggatta gtatagtaaa ttgaaactgt gtcagtccat
     1561 tttttaagag tttaagttca gttcagttta gttcagtgtg agttaatttt tactgctgag
     1621 agtccaaaca ctaaagagca aatccaaata cacaagtcaa taaataaact caatgatggg
     1681 ctaaagatct ggggaaatct gcagatttct gtgcgcacag attctgtgtg ggcctagtca
     1741 ttacctatac tcaacgttta ctctgagaaa cttccatatt ttagaggtaa aatgctcaga
     1801 attgtaaaaa gtcttgtcaa aatacttgga atttgccaaa aacaaaataa aagtcacatc
     1861 acctcattgt ccttggtatc agcaagcact tttaacacaa tcagctgttt ttgtttataa
     1921 ctcccactga aggccatgtg cattactccc aagttacaca gtaaaaagtc acatgtagtt
     1981 tatcatgatc actgctgctc aagcacagct ttatctgtta ttcagtgtgg caatatctga
     2041 aatgtgtgca aaatattatt tattgtagct gatcaccaac actttcagtg tttttttttt
     2101 ctacttttaa tgagaattat gttcttagga tttatcctta taaacctctt taaaaagaaa
     2161 tgttatgtct gcatagacac gacatgaaat tatgaacaga aaatttaact atttaagatc
     2221 aagacttgac atataacatt tctaatgaac attaaacctg aggtaggccc acttgcaaaa
     2281 atgtaactgc tatcactcac tttatacatc attgtgtaac ttgtacacac aaaaatagtg
     2341 aagcaatgtg gcatggaatg caggtcccat tcattcaaag ttattaaagc actttcttaa
     2401 agtgcattac tactgagcta agaccagcta gttgctcata caacatacta caagtagtgt
     2461 catcctaatt tggataaaaa aaatgtccat tttatacatg gaaaagtact cattagatcc
     2521 cccattgttt gtattacggt atttcgtgaa cacaagaggt aaatgaccta cagagctgct
     2581 gttgtgttag attgaaaaca caacacagga tcatggaggc atccatcatt atgacgcatt
     2641 ctgccacttg taactctgca cagacttttt gaaaagttta attgagtctt ggcgttgtcg
     2701 cagaaaaata cgaaaaattc tgtgttaaaa tttgcaaata aaaattccag ttttatagga
     2761 aatcgccatg gccttgttct ttaaccagtt aagcttcccc ttctttcact ctcaagttgc
     2821 aagaagcaag tgtagcaatg tgcacgcgac agccgggtgt gtgacgctgg accaatcaga
     2881 gcacagagct ccgaaagttt accttttatg gctagagccg ggtatgtgcc gtcatataaa
     2941 agagcgcgcc cagcttttca gcctcacttt gagctcctcc acacgcagct agtgcggaat
     3001 atcatctgct tgtaacccat tctcttaagt cgacaacccc ccaaacccaa ggtgagttgg
     3061 ttttacagtt tttaagcgtt taattatagt ttctatctat taatgttaag taaaatgtgt
     3121 aaatggataa atatgatttg atgaagtctt tagacagttc ataacggtct ctttgtagtt
     3181 atctactagg tttgatttat aagtggtggt tcttttgata atctatgtta catttaataa
     3241 atattggctg tatcttaact tttttgtagc gttagtggat gtggcaggtg agaatgtcgg
     3301 taatttaacg tgaccagtcg taggcacgac attgaacatt gaataggccg gtgtgaaatg
     3361 ggtgttaagt cttaatataa agttgtgttc tggctaatct ctctttaaca gccataaatg
     3421 tgatttttaa ctacagtttt gagggtgctg gtgagcatgt tgcacacttg atggatagcc
     3481 ggcatgggaa gttctttgtg caggcagtgc tgcagcaggg tgtgacctac tttagctagc
     3541 cggctaacca gctctcatct gatgtaacgt aaaccccatc aatctaacat cggctcattc
     3601 gtgctttata agatcgaaga tcttgttatt agtaacgggt attggcattt agttttaact
     3661 tggttaattt tctttttgtt atgtgttcta tttaaatgta gcctgcagcc ccctctgtct
     3721 ttaatgaagc tgaagattaa cgttaacgtt agtgaagtgt tgtatccgct atgaaagatt
     3781 tgaattttag aggtgtggtc cactttaata acggctgcgg agttctctgg ctgcacttca
     3841 gccgctgtga ctgtcgaagc agagcccatc agtttttcag gatctcggtg atttttgagg
     3901 ctagactatg aactgaaccg actgagagtg accgtccaga aatgctttag tgtgaagtct
     3961 ccgctcacga ggagtccgtg gactgactgc agatctgtga cgcagtaaca cccggcggca
     4021 gacacccgtt gaaatgcggt tgtgtaattg atcgcaggcg agggtggaag aggatgtgaa
     4081 acttcatttg tgtaaaattt agggagtggt cctggtgtga tgaatgtcga aatctgttcc
     4141 tttttactga gccctacgac tctggctgag tgccacaccg ccggcagccg caaagcggct
     4201 caaaccattg ccttttatgg taataatgag agaatgcaga gggacttcct ttgtctggca
     4261 tatctgaggc gcgcgctgtc actagcgccc accagcggtc agactgtaga atgcagagca
     4321 aaacaggaag ttgactccag atggtcacat gcttgctgaa acgcctctcc ctggcagcag
     4381 tgcaattcta acagtttccc tttcttttac agtggatccg ccaccatggt gagcaagggc
     4441 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     4501 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     4561 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     4621 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     4681 aagtccgcca tgcccgaagg ttggtatcaa agatcttaac tttaaataat gccaattatt
     4741 taaagttacc gaagttccta ttctctagaa agtataggaa cttcgaagtt cctattcttc
     4801 aaaaggtata ggaacttcat aacttcgtat agtacacatt atacgaagtt atataacttc
     4861 gtataggata ctttatacga agttatataa cttcgtatag catacattat acgaagttat
     4921 ggttacaaga caggtttaag gagaccaata gaaactgggc atgtggagac agagaagact
     4981 cttgggtttc tgataggcac tgactctctc tgcctattgg tctattttcc cacccttagg
     5041 gtacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     5101 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     5161 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     5221 tatcatggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat
     5281 cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     5341 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     5401 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     5461 cggcatggac gagctttaca agtaacatat gtaagtatac ttgatatcga catgataaga
     5521 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     5581 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     5641 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     5701 agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga tcagatctgc
     5761 gaagatacgg ccacgggtgc tcttgatcct gtggctgatt ttggactgtg ctgctcgcag
     5821 ctgctgatga atcacatact tcctccattt tcttccactg attgactgtt ataatttccc
     5881 taatttccag gtcaaggtgc tgtgcattgt ggtaatagat gtgacatgac gtcacttcca
     5941 aaggaccaat gaacatgtct gaccaatttc atataatgtg aaaacgattt tcataggcag
     6001 aataaataac atttaaatta aactgggcat cagcgcaatt caattggttt ggtaatagca
     6061 agggaaaata gaatgaagtg atctccaaaa aataagtact ttttgactgt aaataaaatt
     6121 gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat
     6181 tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt aagtacagta
     6241 atcaagtaaa attactcaag tactttacac ctctggttct tgacccccta ccttcagcaa
     6301 gcccagcaga tccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt
     6361 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     6421 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     6481 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     6541 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6601 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     6661 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     6721 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6781 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6841 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6901 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6961 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     7021 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     7081 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     7141 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     7201 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     7261 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7321 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     7381 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     7441 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     7501 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     7561 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     7621 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7681 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7741 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7801 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttg
//
