LOCUS       sequence-158128-        3260 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway ORF Entry clone of human KRAS4B [NM_004985.4 ] with stop 
            codon (for native or N-terminal fusions), G12D/C185S mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3260)
  AUTHORS   Esposito
  TITLE     RAS mutant collection V2.0
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 3260)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "unpublished"
COMMENT     Sequence Label: Hs.KRAS4b G12D/C185S
FEATURES             Location/Qualifiers
     source          1..3260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..45
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     37..54
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     38..54
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    84..183
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(186..218,219..221,222..752)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKSVIM"
     CDS             186..752
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKSVIM"
     protein_bind    complement(751..850)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1227..2018
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2111..2699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2600..2619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2853..2870
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3029..3056)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3148..3234)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaaa
       61 attcgagctc ggtccgagga tccgaaataa tgattttatt ttgactgata gtgacctgtt
      121 cgttgcaaca aattgataag caatgctttt ttataatgcc aactttgtac aaaaaagttg
      181 gcaccatgac tgaatataaa cttgtggtag ttggagctga tggcgtaggc aagagtgcct
      241 tgacgataca gctaattcag aatcattttg tggacgaata tgatccaaca atagaggatt
      301 cctacaggaa gcaagtagta attgatggag aaacctgtct cttggatatt ctcgacacag
      361 caggtcaaga ggagtacagt gcaatgaggg accagtacat gaggactggg gagggctttc
      421 tttgtgtatt tgccataaat aatactaaat catttgaaga tattcaccat tatagagaac
      481 aaattaaaag agttaaggac tctgaagatg tacctatggt cctagtagga aataaatgtg
      541 atttgccttc tagaacagta gacacaaaac aggctcagga cttagcaaga agttatggaa
      601 ttccttttat tgaaacatca gcaaagacaa gacagggtgt tgatgatgcc ttctatacat
      661 tagttcgaga aattcgaaaa cataaagaaa agatgagcaa agatggtaaa aagaagaaaa
      721 agaagtcaaa gacaaagtct gtaattatgt aatcaacttt cttgtacaaa gttggcatta
      781 taggaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa
      841 tcattatttg catgcaagct tagatcctct agaccagcca ggacagaaat gcctcgactt
      901 cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc
      961 agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
     1021 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
     1081 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
     1141 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
     1201 gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc
     1261 aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
     1321 atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg
     1381 ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg
     1441 aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg
     1501 tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat
     1561 ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
     1621 ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg
     1681 aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc
     1741 tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
     1801 gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg
     1861 caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc
     1921 ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact
     1981 acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa
     2041 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     2101 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2161 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2221 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2281 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2341 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2401 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2461 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc
     2521 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2581 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2641 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2701 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2761 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2821 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2881 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2941 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg
     3001 ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     3061 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     3121 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     3181 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
     3241 ggcagttccc tactctcgcg
//
