LOCUS       sequence-257068-        6471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MADR donor plasmid for FlpO+Cre-mediated insertion of oncogenic Hras
            G12V with a TagBFP2 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6471)
  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 6471)
  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 TagBFP2-V5-HRAS G12VWPRE
FEATURES             Location/Qualifiers
     source          1..6471
                     /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)."
     primer_bind     complement(1380..1396)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1381..1397)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             1435..2133
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2143..2184
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(2197..2229,2230..2232,2233..2766)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase H-Ras"
                     /label=H-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     misc_feature    2789..3377
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2842..2862)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3260..3271)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3462..3481)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3527..3582
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3581..3600)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    3922..3955
                     /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(3974..3990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3974..3990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3987..4009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3998..4014
                     /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(4022..4028,4029..4046,4047..4052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4067..4088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4147..4342
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4193..4328
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4348..4482
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4550..4567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4721..5309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4801..4820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5480..6271,6272..6340))
                     /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     6103..6122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6341..6445)
                     /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 ctagcttatc
     1381 gataccgtcg acctcgaggg ggggaattct gcagtcgacg gtaccgccgc caccatgagc
     1441 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     1501 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     1561 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     1621 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     1681 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     1741 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     1801 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     1861 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     1921 aagctcgtgg gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa
     1981 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     2041 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     2101 gacctcccta gcaaactggg gcacaagctt aattccggac tcggcaagcc tatccctaac
     2161 cctctgctgg gcctggacag cacctccgga ctcgagatga cggaatataa gctggtggtg
     2221 gtgggcgccg tcggtgtggg caagagtgcg ctgaccatcc agctgatcca gaaccatttt
     2281 gtggacgaat acgaccccac tatagaggat tcctaccgga agcaggtggt cattgatggg
     2341 gagacgtgcc tgttggacat cctggatacc gccggccagg aggagtacag cgccatgcgg
     2401 gaccagtaca tgcgcaccgg ggagggcttc ctgtgtgtgt ttgccatcaa caacaccaag
     2461 tcttttgagg acatccacca gtacagggag cagatcaaac gggtgaagga ctcggatgac
     2521 gtgcccatgg tgctggtggg gaacaagtgt gacctggctg cacgcactgt ggaatctcgg
     2581 caggctcagg acctcgcccg aagctacggc atcccctaca tcgagacctc ggccaagacc
     2641 cggcagggag tggaggatgc cttctacacg ttggtgcgtg agatccggca gcacaagctg
     2701 cggaagctga accctcctga tgagagtggc cccggctgca tgagctgcaa gtgtgtgctc
     2761 tcctgagcgg ccgcactcct caggtgctaa tcaacctctg gattacaaaa tttgtgaaag
     2821 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     2881 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     2941 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3001 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3061 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3121 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3181 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3241 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3301 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3361 ttgggccgcc tccccgcagg ccgcactcct caggtgcagg ctgcctatca gaaggtggtg
     3421 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa
     3481 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     3541 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     3601 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     3661 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     3721 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     3781 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     3841 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     3901 tggagatccc tcgacctgca ggaagttcct attctctaga aagtatagga acttcaagct
     3961 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4021 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4081 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4141 ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4201 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4261 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4321 tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa
     4381 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4441 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg
     4501 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4561 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4621 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4681 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4741 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4801 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4861 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4921 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4981 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5041 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5101 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5161 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5221 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5281 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5341 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5401 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5461 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5521 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5581 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5641 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5701 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5761 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5821 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5881 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5941 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6001 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6061 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6121 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6181 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6241 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6301 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6361 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6421 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
