LOCUS       sequence-360421-        8041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chimeric AG (N-mApple-intron-C-eGFP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8041)
  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 8041)
  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-AG
FEATURES             Location/Qualifiers
     source          1..8041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    349..382
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    383..416
                     /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    417..450
                     /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(485..518)
                     /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    521..552
                     /label=rox
                     /bound_moiety="Dre recombinase"
     regulatory      569..578
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4076..4238,4239..4250)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     4599..4620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4783..4887
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4888..4956,4957..5748)
                     /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(5106..5125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5919..6507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6408..6427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6661..6678
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6795..6816
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6831..6836,6837..6854,6855..6861)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6869..6885
                     /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     6874..6896
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6893..6909
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6893..6909
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6928..6948
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6930..6948
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     6985..7001
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6985..7001
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(7035..7046,7047..7234)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    7673..7807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7710..7729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7764..7783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7870..7891)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tcgccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtaccc taagggtggg
      241 aaaatagacc aataggcaga gagagtcagt gcctatcaga aacccaagag tcttctctgt
      301 ctccacatgc ccagtttcta ttggtctcct taaacctgtc ttgtaaccat aacttcgtat
      361 aatgtatgct atacgaagtt atataacttc gtataaagta tcctatacga agttatataa
      421 cttcgtataa tgtgtactat acgaagttat gaagttccta taccttttga agaataggaa
      481 cttcgaagtt cctatacttt ctagagaata ggaacttcgg taactttaaa taattggcat
      541 tatttaaagt taagatcttt gataccaacc atggtggcgg atccactgta aaagaaaggg
      601 aaactgttag aattgcactg ctgccaggga gaggcgtttc agcaagcatg tgaccatctg
      661 gagtcaactt cctgttttgc tctgcattct acagtctgac cgctggtggg cgctagtgac
      721 agcgcgcgcc tcagatatgc cagacaaagg aagtccctct gcattctctc attattacca
      781 taaaaggcaa tggtttgagc cgctttgcgg ctgccggcgg tgtggcactc agccagagtc
      841 gtagggctca gtaaaaagga acagatttcg acattcatca caccaggacc actccctaaa
      901 ttttacacaa atgaagtttc acatcctctt ccaccctcgc ctgcgatcaa ttacacaacc
      961 gcatttcaac gggtgtctgc cgccgggtgt tactgcgtca cagatctgca gtcagtccac
     1021 ggactcctcg tgagcggaga cttcacacta aagcatttct ggacggtcac tctcagtcgg
     1081 ttcagttcat agtctagcct caaaaatcac cgagatcctg aaaaactgac gggctctgct
     1141 tcgacagtca cagcggctga agtgcagcca gagaactccg cagccgttat taaagtggac
     1201 cacacctcta aaattcaaaa ctctttcata gcggatacaa tacttcacta acgttaatct
     1261 tcagcttcat taaagacgga gggggctgca ggctacattt aaatagaaca cataacaaaa
     1321 agaaaattaa ccaagttcaa actaaatgcc aatacccgtt actaataaca agatcttcga
     1381 tcttataaag cacgaatggg ccgatgttag attgatgggg tttacgttac atcagatgag
     1441 agctggttag ccggctagct aaagtaggtc acaccctgct gcagcactgc ctgcacaaag
     1501 aacttcccat gccggctatc catcaagtgt gcaacatgct caccagcacc ctcaaaactg
     1561 tagttaaaaa tcacatttat ggctgttgaa gagagattag ccagaacaca actttatatt
     1621 aagacttaac acccatttca caccggccca ttcaatgttc aatgtcgtgc ctacgactgg
     1681 tcacgttaaa ttaccgacat tctcacctgc cacatccact aacgctacaa aaaaagttaa
     1741 gatacagcca atatttatta aatgtaacag atattatcaa aagaaccacc atttataaat
     1801 caaacctagt agataactac aaagagaccg ttatgaactg tctaaagact tcatcaaatc
     1861 atatttatcc atttacacat tttacttaac attaatagat agaaactata attaaacgct
     1921 taaaaactgc aaaaccaact caccttgggt ttggggggtt gtcgacttaa gagaatgggt
     1981 tacaagcaga tgatattccg cactagctgc gtgtggagga gctcaaagtg aggctgagac
     2041 gctgggcgcg ctcttttata tgacggcaca tacccggctc tagccataaa aggtaaactt
     2101 tcggagctct gtgctctgat tggtccagcg tcacacaccc ggctgtcgcg tgcacattgc
     2161 tacacttgct tcttgcaact tgagagtgaa agaaggggaa gcttaactgg ttaaagaaca
     2221 aggccttggc gatttcctat aaaactggaa tttttatttg caaattttaa cacagaattt
     2281 ttcgtatttt tctgcgacaa cgccaagact caattcaacg tttcaaaaag tctgtgcaga
     2341 gttacaagtg gcagaatgcg tcataatgat ggatgcctcc atgatcctgt gttgtgtttt
     2401 caatctaaca caacagcagc tctgtaggtc atttacctct tgtgttcacg aaataccgta
     2461 atacaaacaa tgggggatct aatgagtact tttccatgta taaaatggac atttttttta
     2521 tccaaattag gatgacacta cttgtagtat gttgtaggag caactagctg gtcttagctc
     2581 agtagtaatg cactttaaga aagtgcttta ataactttga atgaatggga cctgcattcc
     2641 atgccacatt gcttcactat ttttgtgtgt acaagttaca caatgatgta taaagtgagt
     2701 gatagcagtt acatttttgc aagtgggcct acctcaggtt taatgttcat tagaaatgtt
     2761 atatgtcaag tcttggtctt aaatagttaa attttctgtt cataatttca tgtcgtgtct
     2821 atgcagacat aacatttctt tttaaagagg tttataagga taaatcctaa gaacataatt
     2881 ctcattaaaa gtagaaaaaa aaacactgaa agtgttggtg atcagctaca ataaataata
     2941 ttttgcacac atttcagatg ttaccacact gaataacaga taaagctgtg cttgagcagc
     3001 agtgatcatg ataaactaca tgtgactttt tactgtgtaa cttgggagta atgcacatgg
     3061 ccttcagtgg gagttataaa caaaaacagc tgattgtgtt aaaagtgctt gctgatacca
     3121 aggacaatga ggtgatgtga cttttatttt gttttcggca aattccaagt attttgacaa
     3181 gactttttac aattctgagc attttgcctc taaaatatgg aagtttctca gagtaaacgt
     3241 tgagtatagg taatgcctag gccctgactg tgcgcacaga aatctgtaga tttccccaga
     3301 tctttagccc atcattgagt ttatttattg acttgtgtat ttggatttgc tctttagtgt
     3361 ttggactctc agcagtaaaa attaacccac actgaactaa actgaactga acttaaactc
     3421 ttaaaaatgg actgacacag tttcaattta ctatactaat cttctaagtt aagctgcttt
     3481 gacacaatct acattgtaaa agcactctag cttgcatgcc tgagaatttc agaatgtaat
     3541 gatacctttg ttagataagg ctgtttacat ctgatagtgg accttaagcc gacacttaaa
     3601 tgataaaaac ggcaaagaat tgcaagtttg atttgcattg gaaagggtcg ctggcttttg
     3661 tgttacacgc cccttatttg tgcttgatta gatgatcccc ctgctcgagg gcccatctgg
     3721 cctgtgtttc agacaccagg gagtctctgc tcacgtttcc tgctatttgc agcctctcta
     3781 tcaagactaa tacacctctt cccgcatcgg ctgcctgtga gaggcttttc agcactgcag
     3841 gattgctttt cagccccaaa agagctaggc ttgacactaa caattttgag aatcagcttc
     3901 tactgaagtt aaatctgagg ttttacaact ttgagtagcg tgtactggca ttagattgtc
     3961 tgtcttatag tttgataatt aaatacaaac agttctaaag caggataaaa ccttgtatgc
     4021 atttcattta atgttttttg agattaaaag cttaaacaag aatctctagt tttctttctt
     4081 gcttttactt ttacttcctt aatactcaag tacaatttta atggagtact tttttacttt
     4141 tactcaagta agattctagc cagatacttt tacttttaat tgagtaaaat tttccctaag
     4201 tacttgtact ttcacttgag taaaattttt gagtactttt tacacctctg tcaagaactc
     4261 ctggacaaac ctctgacctg tgtggaacag agtggatatg ggtgtctgaa cagatattca
     4321 cgtcttttgc agatcagagg gcatttctgg tgttgtgcaa tgacctgggt ccaacaggta
     4381 ggcgtactcg tatccaagag cattgttgga caggaggtag tctccattcc tataaagagg
     4441 aaggaatggc accatgtagt agccgtcatt gtggccaatt ggtgcattgg tacgtgggta
     4501 gatggaccgg ggaggctgat gagttcttag ccagcgtggg ccatcgccct gatagacggt
     4561 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4621 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4681 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4741 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4801 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4861 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4921 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4981 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5041 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5101 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5161 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5221 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5281 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5341 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5401 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5461 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5521 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5581 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5641 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5701 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5761 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5821 gtgaagatcc tttttgataa tctcatgacc aaaatgcctt aacgtgagtt ttcgttccac
     5881 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5941 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6001 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6061 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6121 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6181 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6241 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6301 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6361 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6421 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6481 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6541 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6601 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6661 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     6721 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     6781 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     6841 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     6901 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     6961 gagctccacc gcggtggcgg ccgctctaga actagtggat ctgctgggct tgctgaaggt
     7021 agggggtcaa gaaccagagg tgtaaagtac ttgagtaatt ttacttgatt actgtactta
     7081 agtattattt ttggggattt ttactttact tgagtacaat taaaaatcaa tacttttact
     7141 tttacttaat tacatttttt tagaaaaaaa agtacttttt actccttaca attttattta
     7201 cagtcaaaaa gtacttattt tttggagatc acttcattct attttccctt gctattacca
     7261 aaccaattga attgcgctga tgcccagttt aatttaaatg ttatttattc tgcctatgaa
     7321 aatcgttttc acattatatg aaattggtca gacatgttca ttggtccttt ggaagtgacg
     7381 tcatgtcaca tctattacca caatgcacag caccttgacc tggaaattag ggaaattata
     7441 acagtcaatc agtggaagaa aatggaggaa gtatgtgatt catcagcagc tgcgagcagc
     7501 acagtccaaa atcagccaca ggatcaagag cacccgtggc cgtatcttcg cagatctgat
     7561 ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     7621 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     7681 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     7741 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     7801 cgatatcaag tatacttaca tatgttactt gtaaagctcg tccatgccga gagtgatccc
     7861 ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     7921 cagggcggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     7981 gggggtgttc tgctggtagt ggtcggcgag ctgcacgccg ccgtcctcga tgttgtggcg
     8041 g
//
