LOCUS       sequence-308701-        3855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone encoding EYFP-fused to human KRAS4B, 
            includes a stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3855)
  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 3855)
  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 743 (pDONR221-EYFP-hKRAS4B-WT)
FEATURES             Location/Qualifiers
     source          1..3855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..28)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(120..206)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     255..277
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     269..286
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     270..286
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    302..401
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      404..413
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(410..412,413..415,416..1126)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(455..476)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(716..735)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1063..1084
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1145..1711
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(1713..1812)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1829..1848)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1830..1848)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1853..1869)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1853..1869)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1971..1990)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1982..2791
                     /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(2049..2068)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2938..3526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3427..3446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3680..3697
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg
       61 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga
      121 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
      181 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct
      241 agcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc
      301 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      361 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc taagccacca tggtgagcaa
      421 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
      481 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
      541 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
      601 cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
      661 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
      721 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
      781 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
      841 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
      901 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
      961 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
     1021 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1081 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg gactcagatc
     1141 caccatgact gaatataaac ttgtggtagt tggagctggt ggcgtaggca agagtgcctt
     1201 gacgatacag ctaattcaga atcattttgt ggacgaatat gatccaacaa tagaggattc
     1261 ctacaggaag caagtagtaa ttgatggaga aacctgtctc ttggatattc tcgacacagc
     1321 aggtcaagag gagtacagtg caatgaggga ccagtacatg aggactgggg agggctttct
     1381 ttgtgtattt gccataaata atactaaatc atttgaagat attcaccatt atagagaaca
     1441 aattaaaaga gttaaggact ctgaagatgt acctatggtc ctagtaggaa ataaatgtga
     1501 tttgccttcc agaacagtag acacaaaaca ggctcaggac ttagcaagaa gttatggaat
     1561 tccttttatt gaaacatcag caaagacaag acagggtgtt gatgatgcct tctatacatt
     1621 agttcgagaa attcgaaaac ataaagaaaa gatgagcaaa gatggtaaaa agaagaaaaa
     1681 gaagtcaaag acaaagtgtg taattatgta agacccagct ttcttgtaca aagttggcat
     1741 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     1801 aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc
     1861 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     1921 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     1981 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     2041 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     2101 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     2161 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     2221 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     2281 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2341 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2401 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     2461 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2521 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2581 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     2641 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     2701 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     2761 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     2821 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     2881 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2941 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3001 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3061 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     3121 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3181 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3241 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3301 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3361 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3421 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3481 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3541 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3601 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3661 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     3721 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     3781 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga
     3841 gtttgtagaa acgca
//
