LOCUS       sequence-380468-        8264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockin plasmid with homologous arm for making zMADM-AG zebrafish 
            line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8264)
  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 8264)
  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-AG
FEATURES             Location/Qualifiers
     source          1..8264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     169..188
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    340..373
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    374..407
                     /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    408..441
                     /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(476..509)
                     /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    512..543
                     /label=rox
                     /bound_moiety="Dre recombinase"
     regulatory      560..569
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4290..4452,4453..4464)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     4813..4834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4997..5101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5102..5170,5171..5962)
                     /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(5320..5339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6133..6721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6622..6641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6875..6892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7009..7030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7045..7050,7051..7068,7069..7075)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7083..7099
                     /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     7088..7110
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7107..7123
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7107..7123
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7142..7162
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7144..7162
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     7199..7215
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7199..7215
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(7249..7260,7261..7448)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    7887..8021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7924..7943)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7978..7997
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8084..8105)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ttcgccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag
       61 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
      121 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
      181 ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtacc ctaagggtgg gaaaatagac
      241 caataggcag agagagtcag tgcctatcag aaacccaaga gtcttctctg tctccacatg
      301 cccagtttct attggtctcc ttaaacctgt cttgtaacca taacttcgta taatgtatgc
      361 tatacgaagt tatataactt cgtataaagt atcctatacg aagttatata acttcgtata
      421 atgtgtacta tacgaagtta tgaagttcct ataccttttg aagaatagga acttcgaagt
      481 tcctatactt tctagagaat aggaacttcg gtaactttaa ataattggca ttatttaaag
      541 ttaagatctt tgataccaac catggtggcg gatccactgt aaaagaaagg gaaactgtta
      601 gaattgcact gctgccaggg agaggcgttt cagcaagcat gtgaccatct ggagtcaact
      661 tcctgttttg ctctgcattc tacagtctga ccgctggtgg gcgctagtga cagcgcgcgc
      721 ctcagatatg ccagacaaag gaagtccctc tgcattctct cattattacc ataaaaggca
      781 atggtttgag ccgctttgcg gctgccggcg gtgtggcact cagccagagt cgtagggctc
      841 agtaaaaagg aacagatttc gacattcatc acaccaggac cactccctaa attttacaca
      901 aatgaagttt cacatcctct tccaccctcg cctgcgatca attacacaac cgcatttcaa
      961 cgggtgtctg ccgccgggtg ttactgcgtc acagatctgc agtcagtcca cggactcctc
     1021 gtgagcggag acttcacact aaagcatttc tggacggtca ctctcagtcg gttcagttca
     1081 tagtctagcc tcaaaaatca ccgagatcct gaaaaactga cgggctctgc ttcgacagtc
     1141 acagcggctg aagtgcagcc agagaactcc gcagccgtta ttaaagtgga ccacacctct
     1201 aaaattcaaa actctttcat agcggataca atacttcact aacgttaatc ttcagcttca
     1261 ttaaagacgg agggggctgc aggctacatt taaatagaac acataacaaa aagaaaatta
     1321 accaagttca aactaaatgc caatacccgt tactaataac aagatcttcg atcttataaa
     1381 gcacgaatgg gccgatgtta gattgatggg gtttacgtta catcagatga gagctggtta
     1441 gccggctagc taaagtaggt cacaccctgc tgcagcactg cctgcacaaa gaacttccca
     1501 tgccggctat ccatcaagtg tgcaacatgc tcaccagcac cctcaaaact gtagttaaaa
     1561 atcacattta tggctgttga agagagatta gccagaacac aactttatat taagacttaa
     1621 cacccatttc acaccggccc attcaatgtt caatgtcgtg cctacgactg gtcacgttaa
     1681 attaccgaca ttctcacctg ccacatccac taacgctaca aaaaaagtta agatacagcc
     1741 aatatttatt aaatgtaaca gatattatca aaagaaccac catttataaa tcaaacctag
     1801 tagataacta caaagagacc gttatgaact gtctaaagac ttcatcaaat catatttatc
     1861 catttacaca ttttacttaa cattaataga tagaaactat aattaaacgc ttaaaaactg
     1921 caaaaccaac tcaccttggg tttggggggt tgtcgactta agagaatggg ttacaagcag
     1981 atgatattcc gcactagctg cgtgtggagg agctcaaagt gaggctgaga cgctgggcgc
     2041 gctcttttat atgacggcac atacccggct ctagccataa aaggtaaact ttcggagctc
     2101 tgtgctctga ttggtccagc gtcacacacc cggctgtcgc gtgcacattg ctacacttgc
     2161 ttcttgcaac ttgagagtga aagaagggga agcttaactg gttaaagaac aaggccttgg
     2221 cgatttccta taaaactgga atttttattt gcaaatttta acacagaatt tttcgtattt
     2281 ttctgcgaca acgccaagac tcaattcaac gtttcaaaaa gtctgtgcag agttacaagt
     2341 ggcagaatgc gtcataatga tggatgcctc catgatcctg tgttgtgttt tcaatctaac
     2401 acaacagcag ctctgtaggt catttacctc ttgtgttcac gaaataccgt aatacaaaca
     2461 atgggggatc taatgagtac ttttccatgt ataaaatgga catttttttt atccaaatta
     2521 ggatgacact acttgtagta tgttgtagga gcaactagct ggtcttagct cagtagtaat
     2581 gcactttaag aaagtgcttt aataactttg aatgaatggg acctgcattc catgccacat
     2641 tgcttcacta tttttgtgtg tacaagttac acaatgatgt ataaagtgag tgatagcagt
     2701 tacatttttg caagtgggcc tacctcaggt ttaatgttca ttagaaatgt tatatgtcaa
     2761 gtcttggtct taaatagtta aattttctgt tcataatttc atgtcgtgtc tatgcagaca
     2821 taacatttct ttttaaagag gtttataagg ataaatccta agaacataat tctcattaaa
     2881 agtagaaaaa aaaacactga aagtgttggt gatcagctac aataaataat attttgcaca
     2941 catttcagat gttaccacac tgaataacag ataaagctgt gcttgagcag cagtgatcat
     3001 gataaactac atgtgacttt ttactgtgta acttgggagt aatgcacatg gccttcagtg
     3061 ggagttataa acaaaaacag ctgattgtgt taaaagtgct tgctgatacc aaggacaatg
     3121 aggtgatgtg acttttattt tgttttcggc aaattccaag tattttgaca agacttttta
     3181 caattctgag cattttgcct ctaaaatatg gaagtttctc agagtaaacg ttgagtatag
     3241 gtaatgccta ggccctgact gtgcgcacag aaatctgtag atttccccag atctttagcc
     3301 catcattgag tttatttatt gacttgtgta tttggatttg ctctttagtg tttggactct
     3361 cagcagtaaa aattaaccca cactgaacta aactgaactg aacttaaact cttaaaaatg
     3421 gactgacaca gtttcaattt actatactaa tcttctaagt taagctgctt tgacacaatc
     3481 tacattgtaa aagcactcta gcttgcatgc ctgagaattt cagaatgtaa tgataccttt
     3541 gttagataag gctgtttaca tctgatagtg gaccttaagc cgacacttaa atgataaaaa
     3601 cggcaaagaa ttgcaagttt gatttgcatt ggaaagggtc gctggctttt gtgttacacg
     3661 ccccttattt gtgcttgatt agatgatccc cctgctcgag tttccttaca ggcctaaatt
     3721 tggatttcta atatcacttc accttcctct aaagatactg acagcctaag aaagcacttg
     3781 ggtccagtta agccgcctat tgatcagact tgaagtaaaa atctatttaa tggcctgaaa
     3841 cgcgatcata cattgaaaaa caccaacata agatccgcga aacattgcca aaagcagttt
     3901 tagagagtga aaagtgagaa tgaagatttc agctaaaggt gataaatggt gatgttaatg
     3961 gtgcagaaca gaaggggctc aagttttcaa ccccattaca taagcagtcc aaggctaact
     4021 caacctacca gccatacaaa ttgattgagt aattttgtcc acgaaaggat ctcaccttca
     4081 agccaacctc cagcaaccaa aaataaacta aaaaacctca tttatcttct aactctcact
     4141 ctcacgcttc acatacgctg attggggaga gacttaagag tgttgctcct gcagtcacgt
     4201 taaagcacag ggtggtgaac tcatctggaa atgcatttca tttaatgttt tttgagatta
     4261 aaagcttaaa caagaatctc tagttttctt tcttgctttt acttttactt ccttaatact
     4321 caagtacaat tttaatggag tactttttta cttttactca agtaagattc tagccagata
     4381 cttttacttt taattgagta aaattttccc taagtacttg tactttcact tgagtaaaat
     4441 ttttgagtac tttttacacc tctgtcaaga actcctggac aaacctctga cctgtgtgga
     4501 acagagtgga tatgggtgtc tgaacagata ttcacgtctt ttgcagatca gagggcattt
     4561 ctggtgttgt gcaatgacct gggtccaaca ggtaggcgta ctcgtatcca agagcattgt
     4621 tggacaggag gtagtctcca ttcctataaa gaggaaggaa tggcaccatg tagtagccgt
     4681 cattgtggcc aattggtgca ttggtacgtg ggtagatgga ccggggaggc tgatgagttc
     4741 ttagccagcg tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4801 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4861 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4921 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     4981 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5041 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5101 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5221 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5281 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5341 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5401 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5521 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5641 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5701 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5941 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6001 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6061 gaccaaaatg ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6121 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6181 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6241 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6301 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6361 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6421 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6481 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6541 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6601 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6661 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6721 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6781 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6841 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6901 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6961 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7021 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7081 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     7141 cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc
     7201 tagaactagt ggatctgctg ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa
     7261 gtacttgagt aattttactt gattactgta cttaagtatt atttttgggg atttttactt
     7321 tacttgagta caattaaaaa tcaatacttt tacttttact taattacatt tttttagaaa
     7381 aaaaagtact ttttactcct tacaatttta tttacagtca aaaagtactt attttttgga
     7441 gatcacttca ttctattttc ccttgctatt accaaaccaa ttgaattgcg ctgatgccca
     7501 gtttaattta aatgttattt attctgccta tgaaaatcgt tttcacatta tatgaaattg
     7561 gtcagacatg ttcattggtc ctttggaagt gacgtcatgt cacatctatt accacaatgc
     7621 acagcacctt gacctggaaa ttagggaaat tataacagtc aatcagtgga agaaaatgga
     7681 ggaagtatgt gattcatcag cagctgcgag cagcacagtc caaaatcagc cacaggatca
     7741 agagcacccg tggccgtatc ttcgcagatc tgatctagag gatcataatc agccatacca
     7801 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     7861 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     7921 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     7981 gtttgtccaa actcatcaat gtatcttatc atgtcgatat caagtatact tacatatgtt
     8041 acttgtaaag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     8101 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     8161 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     8221 cgagctgcac gccgccgtcc tcgatgttgt ggcggatctt gaag
//
