LOCUS       sequence-308698-        3964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone encoding V5-tagged mTagBFP2-fused to 
            human KRAS4A with G12D mutation, includes a stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Circ Res. 
            2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500."
COMMENT     Sequence Label: JDW 832 (pDONR221-V5-mTagBFP2-KRAS4A-G12D)
FEATURES             Location/Qualifiers
     source          1..3964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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      complement(3759..3786)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(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
//
