LOCUS       sequence-305005-        6594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MADR donor plasmid for FlpO+Cre- mediated insertion of a SM_FP-FLAG,
            a cytoplasmic fluorescent protein which includes multiple FLAG 
            epitope tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6594)
  AUTHORS   Kim GB, Rincon Fernandez Pacheco D, Saxon D, Yang A, Sabet S, 
            Dutra-Clarke M, Levy R, Watkins A, Park H, Abbasi Akhtar A, Linesch 
            PW, Kobritz N, Chandra SS, Grausam K, Ayala-Sarmiento A, Molina J, 
            Sedivakova K, Hoang K, Tsyporin J, Gareau DS, Filbin MG, Bannykh S, 
            Santiskulvong C, Wang Y, Tang J, Suva ML, Chen B, Danielpour M, 
            Breunig JJ
  TITLE     Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, 
            Stably Integrated Transgenic Elements.
  JOURNAL   Cell. 2019 Sep 19;179(1):251-267.e24. doi: 
            10.1016/j.cell.2019.08.013.
  PUBMED    31539496
REFERENCE   2  (bases 1 to 6594)
  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"; doi: 
            "10.1016/j.cell.2019.08"; journalName: "Cell"; date: "2019-09-19- 
            19"; volume: "179"; issue: "1"; pages: "251-267.e24"
COMMENT     Sequence Label: MADR pDonor smFP-FLAG (dark) WPRE
FEATURES             Location/Qualifiers
     source          1..6594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    32..493
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     polyA_signal    641..775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    895..1029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1149..1283
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1312..1345)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1388..1397
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1397..1420
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1424..1447
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1451..1474
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2003..2026
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2033..2056
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2069..2092
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2099..2122
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2333..2356
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2360..2383
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2387..2410
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     intron          2459..2591
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2574..2593
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2600..3188
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2653..2673)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3071..3082)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3215..3232)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3221..3332
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3428..3448)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3428..3446)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3467..3483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3585..3604)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3650..3705
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3704..3723)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    4045..4078
                     /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)"
     primer_bind     complement(4097..4113)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4121..4137
                     /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(4145..4151,4152..4169,4170..4175))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4190..4211
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4270..4465
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4316..4451
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4471..4605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4673..4690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4844..5432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4924..4943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5603..6394,6395..6463))
                     /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     6226..6245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6464..6568)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagtggatcc gctgtaagtc tgcagaaatt gatgatctat
       61 taaacaataa agatgtccac taaaatggaa gtttttcctg tcatactttg ttaagaaggg
      121 tgagaacaga gtacctacat tttgaatgga aggattggag ctacgggggt gggggtgggg
      181 tgggattaga taaatgcctg ctctttactg aaggctcttt actattgctt tatgataatg
      241 tttcatagtt ggatatcata atttaaacaa gcaaaaccaa attaagggcc agctcattcc
      301 tcccactcat gatctataga tctatagatc tctcgtggga tcattgtttt tctcttgatt
      361 cccactttgt ggttctaagt actgtggttt ccaaatgtgt cagtttcata gcctgaagaa
      421 cgagatcagc agcctctgtt ccacatacac ttcattctca gtattgtttt gccaagttct
      481 aattccatca gaagcttgca gatctgcgac tctagaggat ctgcgactct agaggatcat
      541 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
      601 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
      661 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
      721 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgcga
      781 ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
      841 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
      901 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
      961 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     1021 gtctggatct gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac
     1081 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     1141 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1201 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1261 atgtatctta tcatgtctgg atccccatca agctgatccg gaacccttaa tataacttcg
     1321 tatagcatac attatacgaa gttattaggt ccctcgacct gcagcccaag ctagcgcaat
     1381 tgcggccgcc accatggact acaaggacga cgacgataaa ggagattaca aagatgacga
     1441 tgataagggc gattataagg acgacgatga caagggcggc gtgtcgaaag gcgaggagct
     1501 gttcacaggc gtggtcccca tcctggttga gctggacggt gacgttaacg gccacaaatt
     1561 tagcgtacgt ggtgagggcg agggtgacgc taccaacgga aagctgaccc tgaaattcat
     1621 ctgcacaact ggcaagctgc cggtgccctg gcccacgctg gtaaccactc tgggcggagg
     1681 cgttcaatgt ttcagtcgtt acccggatca catgaagcag catgacttct tcaagtcggc
     1741 catgcccgaa ggctacgtgc aggagcgcac catcagtttt aaggatgacg gcacctacaa
     1801 gactcgcgcc gaggtcaagt tcgaaggaga caccctggtt aatcgtatcg agctcaaagg
     1861 catcgatttc aaggaggacg gaaatattct gggccacaag ctggagtaca attttaacag
     1921 ccacaatgtt tacattactg ccgacaagca gaagaacggc atcaaggcta atttcaagat
     1981 tcgtcacaat gtggagggag gcgactataa ggatgacgat gacaaacaac aagactataa
     2041 ggacgatgat gacaaaggtc agcaaggcga ttacaaggac gatgacgata agcagcaaga
     2101 ttacaaggat gatgatgata agggaggcga cggaagcgtg cagctggcag atcattacca
     2161 acagaacacg cccattggtg acggtccagt cctgctgcct gataaccact acttgtccac
     2221 ccagtcggtg ctgtcgaagg acccgaacga gaagcgtgac cacatggtgc tgctggagtt
     2281 cgtgaccgcc gcaggaatca ccctcggcat ggatgagctt tacaagggcg gagattataa
     2341 agatgacgac gacaagggcg actacaaaga tgatgatgac aagggtgact acaaagacga
     2401 cgatgataaa taaaccggtg ctcgaggtac cgatgaatag ctaaggtcga ggccgcaggt
     2461 aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca
     2521 gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt
     2581 tctctccaca ggtgtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     2641 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     2701 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     2761 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     2821 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     2881 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     2941 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     3001 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     3061 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3121 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3181 ctccccgcct ggaattcgag ctcggtacga tcagcctcga ctgtgccttc tagttgccag
     3241 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3301 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3361 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcccagct
     3421 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     3481 ctgtgtgaaa ttgttatccg ctaattcact cctcaggtgc aggctgccta tcagaaggtg
     3541 gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc
     3601 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     3661 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     3721 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     3781 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     3841 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     3901 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     3961 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     4021 ttatggagat ccctcgacct gcaggaagtt cctattctct agaaagtata ggaacttcaa
     4081 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4141 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4201 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4261 agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4321 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4381 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4441 ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac
     4501 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4561 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa
     4621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4681 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4741 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4801 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4861 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5281 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5341 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5401 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5461 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5521 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5581 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5641 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5701 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5761 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5821 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5881 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5941 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6001 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6061 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6121 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6181 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6241 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6301 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6361 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6421 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6481 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6541 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
