LOCUS Exported 6485 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION MADR donor plasmid for FlpO+Cre-mediated insertion of oncogenic Hras G12V with a TagBFP2 fusion compatible with RCE:loxP mouse strain. ACCESSION . VERSION . KEYWORDS MADR pDonor TagBFP2-V5-HRAS G12V WPRE RCE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6485) 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 6485) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6485 /organism="synthetic DNA construct" /mol_type="other DNA" 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 (GCATACAT)." 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 2197..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 complement(3922..3969) /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." primer_bind complement(3988..4004) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3988..4004) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4001..4023) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4012..4028 /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(4036..4066) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4081..4102 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4161..4356 /label=SV40 promoter /note="SV40 early promoter" rep_origin 4207..4342 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4269..4288 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 4362..4496 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4564..4581) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4735..5323) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4815..4834) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5494..6354) /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 6117..6136 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6355..6459) /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 tataatgtat gctatacgaa 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 atactttcta gagaatagga acttcggaat 3961 aggaacttca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4021 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4081 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4141 cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact 4201 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 4261 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag 4321 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt 4381 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 4441 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct 4501 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4561 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4621 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4681 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4741 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4801 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4861 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4921 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4981 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 5041 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 5101 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5161 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5221 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5281 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5341 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5401 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5461 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5521 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5581 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 5641 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5701 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5761 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5821 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5881 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5941 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6001 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6061 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6121 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6181 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6241 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6301 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6361 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6421 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6481 acctg //