LOCUS       sequence-360419-        8659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of artificial intron separated eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8659)
  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 8659)
  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-GG
FEATURES             Location/Qualifiers
     source          1..8659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    331..364
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    365..398
                     /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    399..432
                     /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(467..500)
                     /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    503..534
                     /label=rox
                     /bound_moiety="Dre recombinase"
     primer_bind     759..780
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      822..831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4329..4491,4492..4503)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     4852..4873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5036..5140
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5141..5209,5210..6001)
                     /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(5359..5378)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6172..6760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6661..6680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6914..6931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7048..7069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7084..7089,7090..7107,7108..7114)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7122..7138
                     /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     7127..7149
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7146..7162
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7146..7162
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7181..7201
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7183..7201
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     7238..7254
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7238..7254
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(7288..7299,7300..7487)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    7926..8060
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7963..7982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8017..8036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8123..8144)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
       61 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
      121 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
      181 ttgaagaaga tggtgcgctc ctggacgtac cctaagggtg ggaaaataga ccaataggca
      241 gagagagtca gtgcctatca gaaacccaag agtcttctct gtctccacat gcccagtttc
      301 tattggtctc cttaaacctg tcttgtaacc ataacttcgt ataatgtatg ctatacgaag
      361 ttatataact tcgtataaag tatcctatac gaagttatat aacttcgtat aatgtgtact
      421 atacgaagtt atgaagttcc tatacctttt gaagaatagg aacttcgaag ttcctatact
      481 ttctagagaa taggaacttc ggtaacttta aataattggc attatttaaa gttaagatct
      541 ttgataccaa ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
      601 gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg gccagggcac
      661 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
      721 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
      781 gatgggcacc accccggtga acagctcctc gcccttgctc accatggtgg cggatccact
      841 gtaaaagaaa gggaaactgt tagaattgca ctgctgccag ggagaggcgt ttcagcaagc
      901 atgtgaccat ctggagtcaa cttcctgttt tgctctgcat tctacagtct gaccgctggt
      961 gggcgctagt gacagcgcgc gcctcagata tgccagacaa aggaagtccc tctgcattct
     1021 ctcattatta ccataaaagg caatggtttg agccgctttg cggctgccgg cggtgtggca
     1081 ctcagccaga gtcgtagggc tcagtaaaaa ggaacagatt tcgacattca tcacaccagg
     1141 accactccct aaattttaca caaatgaagt ttcacatcct cttccaccct cgcctgcgat
     1201 caattacaca accgcatttc aacgggtgtc tgccgccggg tgttactgcg tcacagatct
     1261 gcagtcagtc cacggactcc tcgtgagcgg agacttcaca ctaaagcatt tctggacggt
     1321 cactctcagt cggttcagtt catagtctag cctcaaaaat caccgagatc ctgaaaaact
     1381 gacgggctct gcttcgacag tcacagcggc tgaagtgcag ccagagaact ccgcagccgt
     1441 tattaaagtg gaccacacct ctaaaattca aaactctttc atagcggata caatacttca
     1501 ctaacgttaa tcttcagctt cattaaagac ggagggggct gcaggctaca tttaaataga
     1561 acacataaca aaaagaaaat taaccaagtt caaactaaat gccaataccc gttactaata
     1621 acaagatctt cgatcttata aagcacgaat gggccgatgt tagattgatg gggtttacgt
     1681 tacatcagat gagagctggt tagccggcta gctaaagtag gtcacaccct gctgcagcac
     1741 tgcctgcaca aagaacttcc catgccggct atccatcaag tgtgcaacat gctcaccagc
     1801 accctcaaaa ctgtagttaa aaatcacatt tatggctgtt gaagagagat tagccagaac
     1861 acaactttat attaagactt aacacccatt tcacaccggc ccattcaatg ttcaatgtcg
     1921 tgcctacgac tggtcacgtt aaattaccga cattctcacc tgccacatcc actaacgcta
     1981 caaaaaaagt taagatacag ccaatattta ttaaatgtaa cagatattat caaaagaacc
     2041 accatttata aatcaaacct agtagataac tacaaagaga ccgttatgaa ctgtctaaag
     2101 acttcatcaa atcatattta tccatttaca cattttactt aacattaata gatagaaact
     2161 ataattaaac gcttaaaaac tgcaaaacca actcaccttg ggtttggggg gttgtcgact
     2221 taagagaatg ggttacaagc agatgatatt ccgcactagc tgcgtgtgga ggagctcaaa
     2281 gtgaggctga gacgctgggc gcgctctttt atatgacggc acatacccgg ctctagccat
     2341 aaaaggtaaa ctttcggagc tctgtgctct gattggtcca gcgtcacaca cccggctgtc
     2401 gcgtgcacat tgctacactt gcttcttgca acttgagagt gaaagaaggg gaagcttaac
     2461 tggttaaaga acaaggcctt ggcgatttcc tataaaactg gaatttttat ttgcaaattt
     2521 taacacagaa tttttcgtat ttttctgcga caacgccaag actcaattca acgtttcaaa
     2581 aagtctgtgc agagttacaa gtggcagaat gcgtcataat gatggatgcc tccatgatcc
     2641 tgtgttgtgt tttcaatcta acacaacagc agctctgtag gtcatttacc tcttgtgttc
     2701 acgaaatacc gtaatacaaa caatggggga tctaatgagt acttttccat gtataaaatg
     2761 gacatttttt ttatccaaat taggatgaca ctacttgtag tatgttgtag gagcaactag
     2821 ctggtcttag ctcagtagta atgcacttta agaaagtgct ttaataactt tgaatgaatg
     2881 ggacctgcat tccatgccac attgcttcac tatttttgtg tgtacaagtt acacaatgat
     2941 gtataaagtg agtgatagca gttacatttt tgcaagtggg cctacctcag gtttaatgtt
     3001 cattagaaat gttatatgtc aagtcttggt cttaaatagt taaattttct gttcataatt
     3061 tcatgtcgtg tctatgcaga cataacattt ctttttaaag aggtttataa ggataaatcc
     3121 taagaacata attctcatta aaagtagaaa aaaaaacact gaaagtgttg gtgatcagct
     3181 acaataaata atattttgca cacatttcag atgttaccac actgaataac agataaagct
     3241 gtgcttgagc agcagtgatc atgataaact acatgtgact ttttactgtg taacttggga
     3301 gtaatgcaca tggccttcag tgggagttat aaacaaaaac agctgattgt gttaaaagtg
     3361 cttgctgata ccaaggacaa tgaggtgatg tgacttttat tttgttttcg gcaaattcca
     3421 agtattttga caagactttt tacaattctg agcattttgc ctctaaaata tggaagtttc
     3481 tcagagtaaa cgttgagtat aggtaatgcc taggccctga ctgtgcgcac agaaatctgt
     3541 agatttcccc agatctttag cccatcattg agtttattta ttgacttgtg tatttggatt
     3601 tgctctttag tgtttggact ctcagcagta aaaattaacc cacactgaac taaactgaac
     3661 tgaacttaaa ctcttaaaaa tggactgaca cagtttcaat ttactatact aatcttctaa
     3721 gttaagctgc tttgacacaa tctacattgt aaaagcactc tagcttgcat gcctgagaat
     3781 ttcagaatgt aatgatacct ttgttagata aggctgttta catctgatag tggaccttaa
     3841 gccgacactt aaatgataaa aacggcaaag aattgcaagt ttgatttgca ttggaaaggg
     3901 tcgctggctt ttgtgttaca cgccccttat ttgtgcttga ttagatgatc cccctgctcg
     3961 agggcccatc tggcctgtgt ttcagacacc agggagtctc tgctcacgtt tcctgctatt
     4021 tgcagcctct ctatcaagac taatacacct cttcccgcat cggctgcctg tgagaggctt
     4081 ttcagcactg caggattgct tttcagcccc aaaagagcta ggcttgacac taacaatttt
     4141 gagaatcagc ttctactgaa gttaaatctg aggttttaca actttgagta gcgtgtactg
     4201 gcattagatt gtctgtctta tagtttgata attaaataca aacagttcta aagcaggata
     4261 aaaccttgta tgcatttcat ttaatgtttt ttgagattaa aagcttaaac aagaatctct
     4321 agttttcttt cttgctttta cttttacttc cttaatactc aagtacaatt ttaatggagt
     4381 acttttttac ttttactcaa gtaagattct agccagatac ttttactttt aattgagtaa
     4441 aattttccct aagtacttgt actttcactt gagtaaaatt tttgagtact ttttacacct
     4501 ctgtcaagaa ctcctggaca aacctctgac ctgtgtggaa cagagtggat atgggtgtct
     4561 gaacagatat tcacgtcttt tgcagatcag agggcatttc tggtgttgtg caatgacctg
     4621 ggtccaacag gtaggcgtac tcgtatccaa gagcattgtt ggacaggagg tagtctccat
     4681 tcctataaag aggaaggaat ggcaccatgt agtagccgtc attgtggcca attggtgcat
     4741 tggtacgtgg gtagatggac cggggaggct gatgagttct tagccagcgt gggccatcgc
     4801 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4861 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4921 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4981 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     5041 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5101 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5161 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5221 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5281 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5341 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5401 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5461 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5521 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5581 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5641 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5701 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5761 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5821 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5881 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5941 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6001 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6061 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatgc cttaacgtga
     6121 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6181 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6241 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6301 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6361 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6421 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6481 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6541 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6601 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6661 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6721 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6781 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6841 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6901 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     6961 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     7021 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     7081 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     7141 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
     7201 ggaacaaaag ctggagctcc accgcggtgg cggccgctct agaactagtg gatctgctgg
     7261 gcttgctgaa ggtagggggt caagaaccag aggtgtaaag tacttgagta attttacttg
     7321 attactgtac ttaagtatta tttttgggga tttttacttt acttgagtac aattaaaaat
     7381 caatactttt acttttactt aattacattt ttttagaaaa aaaagtactt tttactcctt
     7441 acaattttat ttacagtcaa aaagtactta ttttttggag atcacttcat tctattttcc
     7501 cttgctatta ccaaaccaat tgaattgcgc tgatgcccag tttaatttaa atgttattta
     7561 ttctgcctat gaaaatcgtt ttcacattat atgaaattgg tcagacatgt tcattggtcc
     7621 tttggaagtg acgtcatgtc acatctatta ccacaatgca cagcaccttg acctggaaat
     7681 tagggaaatt ataacagtca atcagtggaa gaaaatggag gaagtatgtg attcatcagc
     7741 agctgcgagc agcacagtcc aaaatcagcc acaggatcaa gagcacccgt ggccgtatct
     7801 tcgcagatct gatctagagg atcataatca gccataccac atttgtagag gttttacttg
     7861 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     7921 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     7981 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     8041 tatcttatca tgtcgatatc aagtatactt acatatgtta cttgtaaagc tcgtccatgc
     8101 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
     8161 tggggtcttt gctcagggcg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg
     8221 ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg ccgccgtcct
     8281 cgatgttgtg gcggatcttg aagttcgcct tgatgccgtt cttctgcttg tcggccatga
     8341 tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct
     8401 ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca
     8461 cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt
     8521 accctaaggg tgggaaaata gaccaatagg cagagagagt cagtgcctat cagaaaccca
     8581 agagtcttct ctgtctccac atgcccagtt tctattggtc tccttaaacc tgtcttgtaa
     8641 ccataacttc gtataatgt
//
