LOCUS Exported 6471 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. ACCESSION . VERSION . KEYWORDS MADR pDonor TagBFP2-V5-HRAS G12VWPRE 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6471 /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 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 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(4022..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" primer_bind 4255..4274 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" 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(5480..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 //