LOCUS Exported 3964 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Gateway middle entry clone encoding V5-tagged mTagBFP2-fused to human KRAS4A with G12D mutation, includes a stop codon. ACCESSION . VERSION . KEYWORDS JDW 832 (pDONR221-V5-mTagBFP2-KRAS4A-G12D) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3964) AUTHORS Fish JE, Flores-Suarez CP, Boudreau E, Herman AM, Gutierrez MC, Gustafson D, DiStefano PV, Cui M, Chen Z, Berman De Ruiz K, Schexnayder TS, Ward CS, Radovanovic I, Wythe JD TITLE Somatic Gain of KRAS Function in the Endothelium is Sufficient to Cause Vascular Malformations that Require MEK but not PI3K Signaling. JOURNAL Circ Res. 2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500. PUBMED 32552404 REFERENCE 2 (bases 1 to 3964) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3964 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 49..71 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 63..80 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 64..80 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 96..195 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" regulatory 214..223 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 223..264 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 280..990 /codon_start=1 /product="enhanced monomeric blue fluorescent protein (Subach et al., 2011)" /label=mTagBFP2 /note="mammalian codon-optimized" /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV ARYCDLPSKLGHKLN" protein_bind complement(1616..1715) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1732..1751) /label=T7 /note="T7 promoter, forward primer" promoter complement(1733..1751) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1756..1772) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1756..1772) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1874..1893) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1885..2694 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(1952..1971) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 2841..3429 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3330..3349 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3583..3600 /label=L4440 /note="L4440 vector, forward primer" terminator 3759..3786 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 3878..3964 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" ORIGIN 1 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac 61 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga 121 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt 181 acaaaaaagc aggctggatc cactagtgct agcgccacca tgggtaagcc tatccctaac 241 cctctcctcg gtctcgattc tacgggtggt gctggtggta tggtgtctaa gggcgaagag 301 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac 361 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc 421 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc 481 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc 541 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc 601 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg 661 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc 721 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag 781 ctcgtgggcg ggagccatct gatcgcaaac gccaagacca catatagatc caagaaaccc 841 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag 901 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac 961 ctccctagca aactggggca caagcttaat tactcctccg gactcagatc tggaggctcc 1021 ggaggcatga ctgaatataa acttgtggta gttggagctg atggcgtagg caagagtgcc 1081 ttgacgatac agctaattca gaatcatttt gtggacgaat atgatccaac aatagaggat 1141 tcctacagga agcaagtagt aattgatgga gaaacctgtc tcttggatat tctcgacaca 1201 gcaggtcaag aggagtacag tgcaatgagg gaccagtaca tgaggactgg ggagggcttt 1261 ctttgtgtat ttgccataaa taatactaaa tcatttgaag atattcacca ttatagagaa 1321 caaattaaaa gagttaagga ctctgaagat gtacctatgg tcctagtagg aaataaatgt 1381 gatttgcctt ctagaacagt agacacaaaa caggctcagg acttagcaag aagttatgga 1441 attcctttta ttgaaacatc agcaaagaca agacagagag tggaggatgc tttttataca 1501 ttggtgaggg agatccgaca atacagattg aaaaaaatca gcaaagaaga aaagactcct 1561 ggctgtgtga aaattaaaaa atgcattata atgtaaggcg cgccctcgag agatctccca 1621 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa 1681 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt 1741 gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc 1801 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac 1861 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga 1921 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg 1981 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg 2041 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg 2101 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca 2161 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct 2221 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt 2281 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca 2341 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct 2401 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc 2461 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt 2521 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac 2581 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat 2641 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa 2701 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc 2761 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta 2821 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 2881 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 2941 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 3001 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 3061 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 3121 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 3181 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 3241 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 3301 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 3361 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 3421 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 3481 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 3541 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 3601 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 3661 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 3721 acgcgtaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg 3781 ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg 3841 gccgttgctt cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt 3901 tcaccgacaa acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta 3961 tttg //