LOCUS       sequence-362195-        8526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockin plasmid with homologous arm for making zMADM-AA zebrafish 
            line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8526)
  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 8526)
  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-AA
FEATURES             Location/Qualifiers
     source          1..8526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..39)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(193..781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(273..292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(952..1743,1744..1812))
                     /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     1575..1594
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1813..1917)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2080..2101)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(2450..2461,2462..2624)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      6345..6354
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6371..6402
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    6405..6438
                     /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(6473..6506)
                     /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(6507..6540)
                     /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(6541..6574)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             6694..7401
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAF
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNFE
                     DGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALKS
                     EIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     7104..7123
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    7419..7553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7443..7462)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7497..7516
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(7992..8179,8180..8191)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(8225..8241)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8225..8241)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(8278..8298)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8278..8296)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8317..8333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8317..8333)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8330..8352)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8341..8357
                     /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(8365..8371,8372..8389,8390..8395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8410..8431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
       61 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
      121 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
      181 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
      241 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
      301 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
      361 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
      421 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
      481 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
      541 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
      601 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
      661 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
      721 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
      781 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
      841 aggcattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
      901 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
      961 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1021 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1081 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1141 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     1201 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     1261 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     1321 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     1381 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     1441 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     1501 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     1561 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     1621 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     1681 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     1741 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     1801 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     1861 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     1921 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     1981 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2041 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2101 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2161 ccacgctggc taagaactca tcagcctccc cggtccatct acccacgtac caatgcacca
     2221 attggccaca atgacggcta ctacatggtg ccattccttc ctctttatag gaatggagac
     2281 tacctcctgt ccaacaatgc tcttggatac gagtacgcct acctgttgga cccaggtcat
     2341 tgcacaacac cagaaatgcc ctctgatctg caaaagacgt gaatatctgt tcagacaccc
     2401 atatccactc tgttccacac aggtcagagg tttgtccagg agttcttgac agaggtgtaa
     2461 aaagtactca aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa
     2521 ttaaaagtaa aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt
     2581 aaaattgtac ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagattc
     2641 ttgtttaagc ttttaatctc aaaaaacatt aaatgaaatg catttccaga tgagttcacc
     2701 accctgtgct ttaacgtgac tgcaggagca acactcttaa gtctctcccc aatcagcgta
     2761 tgtgaagcgt gagagtgaga gttagaagat aaatgaggtt ttttagttta tttttggttg
     2821 ctggaggttg gcttgaaggt gagatccttt cgtggacaaa attactcaat caatttgtat
     2881 ggctggtagg ttgagttagc cttggactgc ttatgtaatg gggttgaaaa cttgagcccc
     2941 ttctgttctg caccattaac atcaccattt atcaccttta gctgaaatct tcattctcac
     3001 ttttcactct ctaaaactgc ttttggcaat gtttcgcgga tcttatgttg gtgtttttca
     3061 atgtatgatc gcgtttcagg ccattaaata gatttttact tcaagtctga tcaataggcg
     3121 gcttaactgg acccaagtgc tttcttaggc tgtcagtatc tttagaggaa ggtgaagtga
     3181 tattagaaat ccaaatttag gcctgtaagg aaactcgagc agggggatca tctaatcaag
     3241 cacaaataag gggcgtgtaa cacaaaagcc agcgaccctt tccaatgcaa atcaaacttg
     3301 caattctttg ccgtttttat catttaagtg tcggcttaag gtccactatc agatgtaaac
     3361 agccttatct aacaaaggta tcattacatt ctgaaattct caggcatgca agctagagtg
     3421 cttttacaat gtagattgtg tcaaagcagc ttaacttaga agattagtat agtaaattga
     3481 aactgtgtca gtccattttt aagagtttaa gttcagttca gtttagttca gtgtgggtta
     3541 atttttactg ctgagagtcc aaacactaaa gagcaaatcc aaatacacaa gtcaataaat
     3601 aaactcaatg atgggctaaa gatctgggga aatctacaga tttctgtgcg cacagtcagg
     3661 gcctaggcat tacctatact caacgtttac tctgagaaac ttccatattt tagaggcaaa
     3721 atgctcagaa ttgtaaaaag tcttgtcaaa atacttggaa tttgccgaaa acaaaataaa
     3781 agtcacatca cctcattgtc cttggtatca gcaagcactt ttaacacaat cagctgtttt
     3841 tgtttataac tcccactgaa ggccatgtgc attactccca agttacacag taaaaagtca
     3901 catgtagttt atcatgatca ctgctgctca agcacagctt tatctgttat tcagtgtggt
     3961 aacatctgaa atgtgtgcaa aatattattt attgtagctg atcaccaaca ctttcagtgt
     4021 ttttttttct acttttaatg agaattatgt tcttaggatt tatccttata aacctcttta
     4081 aaaagaaatg ttatgtctgc atagacacga catgaaatta tgaacagaaa atttaactat
     4141 ttaagaccaa gacttgacat ataacatttc taatgaacat taaacctgag gtaggcccac
     4201 ttgcaaaaat gtaactgcta tcactcactt tatacatcat tgtgtaactt gtacacacaa
     4261 aaatagtgaa gcaatgtggc atggaatgca ggtcccattc attcaaagtt attaaagcac
     4321 tttcttaaag tgcattacta ctgagctaag accagctagt tgctcctaca acatactaca
     4381 agtagtgtca tcctaatttg gataaaaaaa atgtccattt tatacatgga aaagtactca
     4441 ttagatcccc cattgtttgt attacggtat ttcgtgaaca caagaggtaa atgacctaca
     4501 gagctgctgt tgtgttagat tgaaaacaca acacaggatc atggaggcat ccatcattat
     4561 gacgcattct gccacttgta actctgcaca gactttttga aacgttgaat tgagtcttgg
     4621 cgttgtcgca gaaaaatacg aaaaattctg tgttaaaatt tgcaaataaa aattccagtt
     4681 ttataggaaa tcgccaaggc cttgttcttt aaccagttaa gcttcccctt ctttcactct
     4741 caagttgcaa gaagcaagtg tagcaatgtg cacgcgacag ccgggtgtgt gacgctggac
     4801 caatcagagc acagagctcc gaaagtttac cttttatggc tagagccggg tatgtgccgt
     4861 catataaaag agcgcgccca gcgtctcagc ctcactttga gctcctccac acgcagctag
     4921 tgcggaatat catctgcttg taacccattc tcttaagtcg acaacccccc aaacccaagg
     4981 tgagttggtt ttgcagtttt taagcgttta attatagttt ctatctatta atgttaagta
     5041 aaatgtgtaa atggataaat atgatttgat gaagtcttta gacagttcat aacggtctct
     5101 ttgtagttat ctactaggtt tgatttataa atggtggttc ttttgataat atctgttaca
     5161 tttaataaat attggctgta tcttaacttt ttttgtagcg ttagtggatg tggcaggtga
     5221 gaatgtcggt aatttaacgt gaccagtcgt aggcacgaca ttgaacattg aatgggccgg
     5281 tgtgaaatgg gtgttaagtc ttaatataaa gttgtgttct ggctaatctc tcttcaacag
     5341 ccataaatgt gatttttaac tacagttttg agggtgctgg tgagcatgtt gcacacttga
     5401 tggatagccg gcatgggaag ttctttgtgc aggcagtgct gcagcagggt gtgacctact
     5461 ttagctagcc ggctaaccag ctctcatctg atgtaacgta aaccccatca atctaacatc
     5521 ggcccattcg tgctttataa gatcgaagat cttgttatta gtaacgggta ttggcattta
     5581 gtttgaactt ggttaatttt ctttttgtta tgtgttctat ttaaatgtag cctgcagccc
     5641 cctccgtctt taatgaagct gaagattaac gttagtgaag tattgtatcc gctatgaaag
     5701 agttttgaat tttagaggtg tggtccactt taataacggc tgcggagttc tctggctgca
     5761 cttcagccgc tgtgactgtc gaagcagagc ccgtcagttt ttcaggatct cggtgatttt
     5821 tgaggctaga ctatgaactg aaccgactga gagtgaccgt ccagaaatgc tttagtgtga
     5881 agtctccgct cacgaggagt ccgtggactg actgcagatc tgtgacgcag taacacccgg
     5941 cggcagacac ccgttgaaat gcggttgtgt aattgatcgc aggcgagggt ggaagaggat
     6001 gtgaaacttc atttgtgtaa aatttaggga gtggtcctgg tgtgatgaat gtcgaaatct
     6061 gttccttttt actgagccct acgactctgg ctgagtgcca caccgccggc agccgcaaag
     6121 cggctcaaac cattgccttt tatggtaata atgagagaat gcagagggac ttcctttgtc
     6181 tggcatatct gaggcgcgcg ctgtcactag cgcccaccag cggtcagact gtagaatgca
     6241 gagcaaaaca ggaagttgac tccagatggt cacatgcttg ctgaaacgcc tctccctggc
     6301 agcagtgcaa ttctaacagt ttccctttct tttacagtgg atccgccacc atggttggta
     6361 tcaaagatct taactttaaa taatgccaat tatttaaagt taccgaagtt cctattctct
     6421 agaaagtata ggaacttcga agttcctatt cttcaaaagg tataggaact tcataacttc
     6481 gtatagtaca cattatacga agttatataa cttcgtatag gatactttat acgaagttat
     6541 ataacttcgt atagcataca ttatacgaag ttatggttac aagacaggtt taaggagacc
     6601 aatagaaact gggcatgtgg agacagagaa gactcttggg tttctgatag gcactgactc
     6661 tctctgccta ttggtctatt ttcccaccct taggtgagca agggcgagga gaataacatg
     6721 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     6781 gagttcgaga tcgagggcga gggcgagggc cgcccctacg aggcctttca gaccgctaag
     6841 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     6901 atgtacggct ccaaggtcta cattaagcac ccagccgaca tccccgacta cttcaagctg
     6961 tccttccccg agggcttcag gtgggagcgc gtgatgaact tcgaggacgg cggcattatt
     7021 cacgttaacc aggactcctc cctgcaggac ggcgtgttca tctacaaggt gaagctgcgc
     7081 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     7141 tccgaggagc ggatgtaccc cgaggacggc gccctgaaga gcgagatcaa gaagaggctg
     7201 aagctgaagg acggcggcca ctacgccgcc gaggtcaaga ccacctacaa ggccaagaag
     7261 cccgtgcagc tgcccggcgc ctacatcgtc gacatcaagt tggacatcgt gtcccacaac
     7321 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     7381 atggacgagc tgtacaagta acatatgtaa gtatacttga tatcgacatg ataagataca
     7441 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     7501 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     7561 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     7621 agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctagatcag atctgcgaag
     7681 atacggccac gggtgctctt gatcctgtgg ctgattttgg actgtgctgc tcgcagctgc
     7741 tgatgaatca catacttcct ccattttctt ccactgattg actgttataa tttccctaat
     7801 ttccaggtca aggtgctgtg cattgtggta atagatgtga catgacgtca cttccaaagg
     7861 accaatgaac atgtctgacc aatttcatat aatgtgaaaa cgattttcat aggcagaata
     7921 aataacattt aaattaaact gggcatcagc gcaattcaat tggtttggta atagcaaggg
     7981 aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa
     8041 ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat
     8101 ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca
     8161 agtaaaatta ctcaagtact ttacacctct ggttcttgac cccctacctt cagcaagccc
     8221 agcagatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     8281 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     8341 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     8401 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     8461 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     8521 tttgcg
//
