LOCUS       sequence-308696-        3861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone encoding EGFP-fused to human KRAS4B with 
            G12V mutation, includes a stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3861)
  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 3861)
  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 742 (pDONR221-EGFP-KRAS4B-G12V)
FEATURES             Location/Qualifiers
     source          1..3861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(92..178)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     227..249
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     241..258
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     242..258
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    274..373
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      376..385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(382..384,385..387,388..1098)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(382..384,385..387,388..1098)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(427..448)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(688..707)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1035..1056
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(1123..1155,1156..1158,1159..1689)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(1691..1790)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1807..1826)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1808..1826)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1831..1847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1831..1847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1949..1968)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1960..2769
                     /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(2027..2046)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2916..3504
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3405..3424
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3658..3675
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3834..3861)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
       61 gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
      121 acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat
      181 gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt
      241 tgtaaaacga cggccagtct taagctcggg ccccaaataa tgattttatt ttgactgata
      301 gtgacctgtt cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtac
      361 aaaaaagcag gctaagccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      421 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      481 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      541 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      601 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      661 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      721 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      781 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
      841 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
      901 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      961 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1021 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1081 atggacgagc tgtacaagta ctcagatctc gagctcaagc ttatgactga atataaactt
     1141 gtggtagttg gagctgttgg cgtaggcaag agtgccttga cgatacagct aattcagaat
     1201 cattttgtgg acgaatatga tccaacaata gaggattcct acaggaagca agtagtaatt
     1261 gatggagaaa cctgtctctt ggatattctc gacacagcag gtcaagagga gtacagtgca
     1321 atgagggacc agtacatgag gactggggag ggctttcttt gtgtatttgc cataaataat
     1381 actaaatcat ttgaagatat tcaccattat agagaacaaa ttaaaagagt taaggactct
     1441 gaagatgtac ctatggtcct agtaggaaat aaatgtgatt tgccttccag aacagtagac
     1501 acaaaacagg ctcaggactt agcaagaagt tatggaattc cttttattga aacatcagca
     1561 aagacaagac agggtgttga tgatgccttc tatacattag ttcgagaaat tcgaaaacat
     1621 aaagaaaaga tgagcaaaga tggtaaaaag aagaaaaaga agtcaaagac aaagtgtgta
     1681 attatgtaag acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     1741 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     1801 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     1861 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     1921 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     1981 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     2041 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     2101 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     2161 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     2221 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     2281 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2341 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2401 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2461 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2521 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2581 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2641 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2701 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2761 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     2821 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     2881 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2941 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3001 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3061 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3121 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3181 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3241 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3301 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3361 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3421 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3481 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3541 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3601 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3661 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     3721 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3781 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
     3841 catccgtcag gatggccttc t
//
