LOCUS       sequence-380465-        8797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockin plasmid with homologous arm for making zMADM-GA zebrafish 
            line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8797)
  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 8797)
  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-GA
FEATURES             Location/Qualifiers
     source          1..8797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..37)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(191..779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(271..290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(950..1741,1742..1810))
                     /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     1573..1592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1811..1915)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2078..2099)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(2448..2459,2460..2622)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      6343..6352
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6394..6415)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    6640..6671
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    6674..6707
                     /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(6742..6775)
                     /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(6776..6809)
                     /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(6810..6843)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             6963..7670
                     /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     7373..7392
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    7688..7822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7712..7731)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7766..7785
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(8261..8448,8449..8460)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(8494..8510)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8494..8510)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(8547..8567)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8547..8565)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8586..8602)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8586..8602)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8599..8621)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8610..8626
                     /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(8634..8640,8641..8658,8659..8664))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8679..8700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
       61 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
      121 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
      181 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
      241 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
      301 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
      361 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
      421 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      481 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      541 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      601 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
      661 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
      721 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
      781 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
      841 gcattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
      901 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      961 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     1021 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     1081 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     1141 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     1201 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     1261 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     1321 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     1381 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     1441 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     1501 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     1561 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     1621 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     1681 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     1741 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     1801 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1861 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     1921 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1981 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2041 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2101 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2161 acgctggcta agaactcatc agcctccccg gtccatctac ccacgtacca atgcaccaat
     2221 tggccacaat gacggctact acatggtgcc attccttcct ctttatagga atggagacta
     2281 cctcctgtcc aacaatgctc ttggatacga gtacgcctac ctgttggacc caggtcattg
     2341 cacaacacca gaaatgccct ctgatctgca aaagacgtga atatctgttc agacacccat
     2401 atccactctg ttccacacag gtcagaggtt tgtccaggag ttcttgacag aggtgtaaaa
     2461 agtactcaaa aattttactc aagtgaaagt acaagtactt agggaaaatt ttactcaatt
     2521 aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa gtaaaaaagt actccattaa
     2581 aattgtactt gagtattaag gaagtaaaag taaaagcaag aaagaaaact agagattctt
     2641 gtttaagctt ttaatctcaa aaaacattaa atgaaatgca tttccagatg agttcaccac
     2701 cctgtgcttt aacgtgactg caggagcaac attcttaagt ctctccccaa tcagcgtatg
     2761 tgaagcgtga gagtgagagt tagaagataa atgaggtttt ttagtttatt tttggttgct
     2821 ggaggttggc ttgaaggtga gatcctttcg tggacaaaat tactcaatca atttgtatgg
     2881 ctggtaggtt gagttagcct tggactgctt atgtaatggg gttgaaaact tgagcccctt
     2941 ctgttctgca ccattaacat caccatttat cacctttagc tgaaatcttc attctcactt
     3001 ttcactctct aaaactgctt ttggcaatgt ttcgcggatc ttatgttggt gtttttcaat
     3061 gtatgatcgc gtttcaggcc attaaatgga tttttacttc aagtctgatc aataggcggc
     3121 ttaactggac ccaagtgctt tcttaggctg tcagtatctt tagaggaagg tgaagtgata
     3181 ttagaaatcc aaatttaggc ctgtaaggaa actcgagcag ggggatcatc taatcaagca
     3241 caaataaggg gcgtgtaaca caaaagccag cgaccctttc caatgcaaat caaacttgca
     3301 attctttgcc gtttttatca tttaagtgtc ggcttaaggt ccactatcag atgtaaacag
     3361 ccttatctaa caaaggtatc attacattct gaaattctca ggcatgcaag ctagagtgct
     3421 tttacaatgt agattgtgtc aaagcagctt aacttagaag attagtatag taaattgaaa
     3481 ctgtgtcagt ccatttttaa gagtttaagt tcagttcagt ttagttcagt gtgggttaat
     3541 ttttactgct gagagtccaa acactaaaga gcaaatccaa atacacaagt caataaataa
     3601 actcaatgat gggctaaaga tctggggaaa tctacagatt tctgtgcgca cagtcagggc
     3661 ctaggcatta cctatactca acgtttactc tgagaaactt ccatatttta gaggcaaaat
     3721 gctcagaatt gtaaaaagtc ttgtcaaaat acttggaatt tgccgaaaac aaaataaaag
     3781 tcacatcacc tcattgtcct tggtatcagc aagcactttt aacacaatca gctgtttttg
     3841 tttataactc ccactgaagg ccatgtgcat tactcccaag ttacacagta aaaagtcaca
     3901 tgtagtttat catgatcact gctgctcaag cacagcttta tctgttattc agtgtggtaa
     3961 catctgaaat gtgtgcaaaa tattatttat tgtagctgat caccaacact ttcagtgttt
     4021 ttttttctac ttttaatgag aattatgttc ttaggattta tccttataaa cctctttaaa
     4081 aagaaatgtt atgtctgcat agacacgaca tgaaattatg aacagaaaat ttaactattt
     4141 aagaccaaga cttgacatat aacatttcta atgaacatta aacctgaggt aggcccactt
     4201 gcaaaaatgt aactgctatc actcacttta tacatcattg tgtaacttgt acacacaaaa
     4261 atagtgaagc aatgtggcat ggaatgcagg tcccattcat tcaaagttat taaagcactt
     4321 tcttaaagtg cattactact gagctaagac cagctagttg ctcctacaac atactacaag
     4381 tagtgtcatc ctaatttgga taaaaaaaat gtccatttta tacatggaaa agtactcatt
     4441 agatccccca ttgtttgtat tacggtattt cgtgaacaca agaggtaaat gacctacaga
     4501 gctgctgttg tgttagattg aaaacacaac acaggatcat ggaggcatcc atcattatga
     4561 cgcattctgc cacttgtaac tctgcacaga ctttttgaaa cgttgaattg agtcttggcg
     4621 ttgtcgcaga aaaatacgaa aaattctgtg ttaaaatttg caaataaaaa ttccagtttt
     4681 ataggaaatc gccaaggcct tgttctttaa ccagttaagc ttccccttct ttcactctca
     4741 agttgcaaga agcaagtgta gcaatgtgca cgcgacagcc gggtgtgtga cgctggacca
     4801 atcagagcac agagctccga aagtttacct tttatggcta gagccgggta tgtgccgtca
     4861 tataaaagag cgcgcccagc gtctcagcct cactttgagc tcctccacac gcagctagtg
     4921 cggaatatca tctgcttgta acccattctc ttaagtcgac aaccccccaa acccaaggtg
     4981 agttggtttt gcagttttta agcgtttaat tatagtttct atctattaat gttaagtaaa
     5041 atgtgtaaat ggataaatat gatttgatga agtctttaga cagttcataa cggtctcttt
     5101 gtagttatct actaggtttg atttataaat ggtggttctt ttgataatat ctgttacatt
     5161 taataaatat tggctgtatc ttaacttttt ttgtagcgtt agtggatgtg gcaggtgaga
     5221 atgtcggtaa tttaacgtga ccagtcgtag gcacgacatt gaacattgaa tgggccggtg
     5281 tgaaatgggt gttaagtctt aatataaagt tgtgttctgg ctaatctctc ttcaacagcc
     5341 ataaatgtga tttttaacta cagttttgag ggtgctggtg agcatgttgc acacttgatg
     5401 gatagccggc atgggaagtt ctttgtgcag gcagtgctgc agcagggtgt gacctacttt
     5461 agctagccgg ctaaccagct ctcatctgat gtaacgtaaa ccccatcaat ctaacatcgg
     5521 cccattcgtg ctttataaga tcgaagatct tgttattagt aacgggtatt ggcatttagt
     5581 ttgaacttgg ttaattttct ttttgttatg tgttctattt aaatgtagcc tgcagccccc
     5641 tccgtcttta atgaagctga agattaacgt tagtgaagta ttgtatccgc tatgaaagag
     5701 ttttgaattt tagaggtgtg gtccacttta ataacggctg cggagttctc tggctgcact
     5761 tcagccgctg tgactgtcga agcagagccc gtcagttttt caggatctcg gtgatttttg
     5821 aggctagact atgaactgaa ccgactgaga gtgaccgtcc agaaatgctt tagtgtgaag
     5881 tctccgctca cgaggagtcc gtggactgac tgcagatctg tgacgcagta acacccggcg
     5941 gcagacaccc gttgaaatgc ggttgtgtaa ttgatcgcag gcgagggtgg aagaggatgt
     6001 gaaacttcat ttgtgtaaaa tttagggagt ggtcctggtg tgatgaatgt cgaaatctgt
     6061 tcctttttac tgagccctac gactctggct gagtgccaca ccgccggcag ccgcaaagcg
     6121 gctcaaacca ttgcctttta tggtaataat gagagaatgc agagggactt cctttgtctg
     6181 gcatatctga ggcgcgcgct gtcactagcg cccaccagcg gtcagactgt agaatgcaga
     6241 gcaaaacagg aagttgactc cagatggtca catgcttgct gaaacgcctc tccctggcag
     6301 cagtgcaatt ctaacagttt ccctttcttt tacagtggat ccgccaccat ggtgagcaag
     6361 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     6421 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     6481 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     6541 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     6601 ttcaagtccg ccatgcccga aggttggtat caaagatctt aactttaaat aatgccaatt
     6661 atttaaagtt accgaagttc ctattctcta gaaagtatag gaacttcgaa gttcctattc
     6721 ttcaaaaggt ataggaactt cataacttcg tatagtacac attatacgaa gttatataac
     6781 ttcgtatagg atactttata cgaagttata taacttcgta tagcatacat tatacgaagt
     6841 tatggttaca agacaggttt aaggagacca atagaaactg ggcatgtgga gacagagaag
     6901 actcttgggt ttctgatagg cactgactct ctctgcctat tggtctattt tcccaccctt
     6961 aggtgagcaa gggcgaggag aataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     7021 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     7081 gcccctacga ggcctttcag accgctaagc tgaaggtgac caagggtggc cccctgccct
     7141 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggtctac attaagcacc
     7201 cagccgacat ccccgactac ttcaagctgt ccttccccga gggcttcagg tgggagcgcg
     7261 tgatgaactt cgaggacggc ggcattattc acgttaacca ggactcctcc ctgcaggacg
     7321 gcgtgttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     7381 tgcagaagaa gaccatgggc tgggaggcct ccgaggagcg gatgtacccc gaggacggcg
     7441 ccctgaagag cgagatcaag aagaggctga agctgaagga cggcggccac tacgccgccg
     7501 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacatcgtcg
     7561 acatcaagtt ggacatcgtg tcccacaacg aggactacac catcgtggaa cagtacgaac
     7621 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa catatgtaag
     7681 tatacttgat atcgacatga taagatacat tgatgagttt ggacaaacca caactagaat
     7741 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     7801 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     7861 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga
     7921 ttatgatcct ctagatcaga tctgcgaaga tacggccacg ggtgctcttg atcctgtggc
     7981 tgattttgga ctgtgctgct cgcagctgct gatgaatcac atacttcctc cattttcttc
     8041 cactgattga ctgttataat ttccctaatt tccaggtcaa ggtgctgtgc attgtggtaa
     8101 tagatgtgac atgacgtcac ttccaaagga ccaatgaaca tgtctgacca atttcatata
     8161 atgtgaaaac gattttcata ggcagaataa ataacattta aattaaactg ggcatcagcg
     8221 caattcaatt ggtttggtaa tagcaaggga aaatagaatg aagtgatctc caaaaaataa
     8281 gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa
     8341 tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc
     8401 ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg
     8461 gttcttgacc ccctaccttc agcaagccca gcagatccac tagttctaga gcggccgcca
     8521 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     8581 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     8641 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     8701 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     8761 tgaatcggcc aacgcgcggg gagaggcggt ttgcgta
//
