LOCUS       sequence-359394-        8303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of artificial intron separated mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8303)
  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 8303)
  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: pTol2-eab2-AA
FEATURES             Location/Qualifiers
     source          1..8303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(197..301)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(464..485)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(834..845,846..1008)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      4506..4515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4532..4563
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    4566..4599
                     /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(4634..4667)
                     /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(4668..4701)
                     /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(4702..4735)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4855..5562
                     /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     5265..5284
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5580..5714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5604..5623)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5658..5677
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(6153..6340,6341..6352)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6386..6402)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6386..6402)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6439..6459)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6439..6457)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6478..6494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6478..6494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6491..6513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6502..6518
                     /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(6526..6532,6533..6550,6551..6556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6571..6592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6709..6726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6880..7468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6960..6979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7639..8303,1..127,128..196))
                     /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     8262..8281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
       61 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
      121 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
      181 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
      241 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
      301 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
      361 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
      421 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
      481 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
      541 tggcccacgc tggctaagaa ctcatcagcc tccccggtcc atctacccac gtaccaatgc
      601 accaattggc cacaatgacg gctactacat ggtgccattc cttcctcttt ataggaatgg
      661 agactacctc ctgtccaaca atgctcttgg atacgagtac gcctacctgt tggacccagg
      721 tcattgcaca acaccagaaa tgccctctga tctgcaaaag acgtgaatat ctgttcagac
      781 acccatatcc actctgttcc acacaggtca gaggtttgtc caggagttct tgacagaggt
      841 gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa gtacttaggg aaaattttac
      901 tcaattaaaa gtaaaagtat ctggctagaa tcttacttga gtaaaagtaa aaaagtactc
      961 cattaaaatt gtacttgagt attaaggaag taaaagtaaa agcaagaaag aaaactagag
     1021 attcttgttt aagcttttaa tctcaaaaaa cattaaatga aatgcataca aggttttatc
     1081 ctgctttaga actgtttgta tttaattatc aaactataag acagacaatc taatgccagt
     1141 acacgctact caaagttgta aaacctcaga tttaacttca gtagaagctg attctcaaaa
     1201 ttgttagtgt caagcctagc tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc
     1261 ctctcacagg cagccgatgc gggaagaggt gtattagtct tgatagagag gctgcaaata
     1321 gcaggaaacg tgagcagaga ctccctggtg tctgaaacac aggccagatg ggccctcgag
     1381 cagggggatc atctaatcaa gcacaaataa ggggcgtgta acacaaaagc cagcgaccct
     1441 ttccaatgca aatcaaactt gcaattcttt gccgttttta tcatttaagt gtcggcttaa
     1501 ggtccactat cagatgtaaa cagccttatc taacaaaggt atcattacat tctgaaattc
     1561 tcaggcatgc aagctagagt gcttttacaa tgtagattgt gtcaaagcag cttaacttag
     1621 aagattagta tagtaaattg aaactgtgtc agtccatttt taagagttta agttcagttc
     1681 agtttagttc agtgtgggtt aatttttact gctgagagtc caaacactaa agagcaaatc
     1741 caaatacaca agtcaataaa taaactcaat gatgggctaa agatctgggg aaatctacag
     1801 atttctgtgc gcacagtcag ggcctaggca ttacctatac tcaacgttta ctctgagaaa
     1861 cttccatatt ttagaggcaa aatgctcaga attgtaaaaa gtcttgtcaa aatacttgga
     1921 atttgccgaa aacaaaataa aagtcacatc acctcattgt ccttggtatc agcaagcact
     1981 tttaacacaa tcagctgttt ttgtttataa ctcccactga aggccatgtg cattactccc
     2041 aagttacaca gtaaaaagtc acatgtagtt tatcatgatc actgctgctc aagcacagct
     2101 ttatctgtta ttcagtgtgg taacatctga aatgtgtgca aaatattatt tattgtagct
     2161 gatcaccaac actttcagtg tttttttttc tacttttaat gagaattatg ttcttaggat
     2221 ttatccttat aaacctcttt aaaaagaaat gttatgtctg catagacacg acatgaaatt
     2281 atgaacagaa aatttaacta tttaagacca agacttgaca tataacattt ctaatgaaca
     2341 ttaaacctga ggtaggccca cttgcaaaaa tgtaactgct atcactcact ttatacatca
     2401 ttgtgtaact tgtacacaca aaaatagtga agcaatgtgg catggaatgc aggtcccatt
     2461 cattcaaagt tattaaagca ctttcttaaa gtgcattact actgagctaa gaccagctag
     2521 ttgctcctac aacatactac aagtagtgtc atcctaattt ggataaaaaa aatgtccatt
     2581 ttatacatgg aaaagtactc attagatccc ccattgtttg tattacggta tttcgtgaac
     2641 acaagaggta aatgacctac agagctgctg ttgtgttaga ttgaaaacac aacacaggat
     2701 catggaggca tccatcatta tgacgcattc tgccacttgt aactctgcac agactttttg
     2761 aaacgttgaa ttgagtcttg gcgttgtcgc agaaaaatac gaaaaattct gtgttaaaat
     2821 ttgcaaataa aaattccagt tttataggaa atcgccaagg ccttgttctt taaccagtta
     2881 agcttcccct tctttcactc tcaagttgca agaagcaagt gtagcaatgt gcacgcgaca
     2941 gccgggtgtg tgacgctgga ccaatcagag cacagagctc cgaaagttta ccttttatgg
     3001 ctagagccgg gtatgtgccg tcatataaaa gagcgcgccc agcgtctcag cctcactttg
     3061 agctcctcca cacgcagcta gtgcggaata tcatctgctt gtaacccatt ctcttaagtc
     3121 gacaaccccc caaacccaag gtgagttggt tttgcagttt ttaagcgttt aattatagtt
     3181 tctatctatt aatgttaagt aaaatgtgta aatggataaa tatgatttga tgaagtcttt
     3241 agacagttca taacggtctc tttgtagtta tctactaggt ttgatttata aatggtggtt
     3301 cttttgataa tatctgttac atttaataaa tattggctgt atcttaactt tttttgtagc
     3361 gttagtggat gtggcaggtg agaatgtcgg taatttaacg tgaccagtcg taggcacgac
     3421 attgaacatt gaatgggccg gtgtgaaatg ggtgttaagt cttaatataa agttgtgttc
     3481 tggctaatct ctcttcaaca gccataaatg tgatttttaa ctacagtttt gagggtgctg
     3541 gtgagcatgt tgcacacttg atggatagcc ggcatgggaa gttctttgtg caggcagtgc
     3601 tgcagcaggg tgtgacctac tttagctagc cggctaacca gctctcatct gatgtaacgt
     3661 aaaccccatc aatctaacat cggcccattc gtgctttata agatcgaaga tcttgttatt
     3721 agtaacgggt attggcattt agtttgaact tggttaattt tctttttgtt atgtgttcta
     3781 tttaaatgta gcctgcagcc ccctccgtct ttaatgaagc tgaagattaa cgttagtgaa
     3841 gtattgtatc cgctatgaaa gagttttgaa ttttagaggt gtggtccact ttaataacgg
     3901 ctgcggagtt ctctggctgc acttcagccg ctgtgactgt cgaagcagag cccgtcagtt
     3961 tttcaggatc tcggtgattt ttgaggctag actatgaact gaaccgactg agagtgaccg
     4021 tccagaaatg ctttagtgtg aagtctccgc tcacgaggag tccgtggact gactgcagat
     4081 ctgtgacgca gtaacacccg gcggcagaca cccgttgaaa tgcggttgtg taattgatcg
     4141 caggcgaggg tggaagagga tgtgaaactt catttgtgta aaatttaggg agtggtcctg
     4201 gtgtgatgaa tgtcgaaatc tgttcctttt tactgagccc tacgactctg gctgagtgcc
     4261 acaccgccgg cagccgcaaa gcggctcaaa ccattgcctt ttatggtaat aatgagagaa
     4321 tgcagaggga cttcctttgt ctggcatatc tgaggcgcgc gctgtcacta gcgcccacca
     4381 gcggtcagac tgtagaatgc agagcaaaac aggaagttga ctccagatgg tcacatgctt
     4441 gctgaaacgc ctctccctgg cagcagtgca attctaacag tttccctttc ttttacagtg
     4501 gatccgccac catggttggt atcaaagatc ttaactttaa ataatgccaa ttatttaaag
     4561 ttaccgaagt tcctattctc tagaaagtat aggaacttcg aagttcctat tcttcaaaag
     4621 gtataggaac ttcataactt cgtatagtac acattatacg aagttatata acttcgtata
     4681 ggatacttta tacgaagtta tataacttcg tatagcatac attatacgaa gttatggtta
     4741 caagacaggt ttaaggagac caatagaaac tgggcatgtg gagacagaga agactcttgg
     4801 gtttctgata ggcactgact ctctctgcct attggtctat tttcccaccc ttaggtgagc
     4861 aagggcgagg agaataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     4921 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     4981 gaggcctttc agaccgctaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     5041 gacatcctgt cccctcagtt catgtacggc tccaaggtct acattaagca cccagccgac
     5101 atccccgact acttcaagct gtccttcccc gagggcttca ggtgggagcg cgtgatgaac
     5161 ttcgaggacg gcggcattat tcacgttaac caggactcct ccctgcagga cggcgtgttc
     5221 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     5281 aagaccatgg gctgggaggc ctccgaggag cggatgtacc ccgaggacgg cgccctgaag
     5341 agcgagatca agaagaggct gaagctgaag gacggcggcc actacgccgc cgaggtcaag
     5401 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacatcgt cgacatcaag
     5461 ttggacatcg tgtcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     5521 ggccgccact ccaccggcgg catggacgag ctgtacaagt aacatatgta agtatacttg
     5581 atatcgacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     5641 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     5701 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     5761 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc
     5821 ctctagatca gatctgcgaa gatacggcca cgggtgctct tgatcctgtg gctgattttg
     5881 gactgtgctg ctcgcagctg ctgatgaatc acatacttcc tccattttct tccactgatt
     5941 gactgttata atttccctaa tttccaggtc aaggtgctgt gcattgtggt aatagatgtg
     6001 acatgacgtc acttccaaag gaccaatgaa catgtctgac caatttcata taatgtgaaa
     6061 acgattttca taggcagaat aaataacatt taaattaaac tgggcatcag cgcaattcaa
     6121 ttggtttggt aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt
     6181 tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta
     6241 agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat
     6301 aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga
     6361 ccccctacct tcagcaagcc cagcagatcc actagttcta gagcggccgc caccgcggtg
     6421 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     6481 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     6541 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     6601 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6661 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6721 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6781 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6841 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6901 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6961 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     7021 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     7081 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7141 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     7201 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7261 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7321 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7381 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7441 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     7501 cgctcagtgg aacgaaaact cacgttaagg cattttggtc atgagattat caaaaaggat
     7561 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     7621 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7681 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7741 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7801 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7861 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7921 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7981 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     8041 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     8101 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     8161 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     8221 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8281 cagaacttta aaagtgctca tca
//
