LOCUS       sequence-213454-        4533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses activated human KRAS G12C (amino acids 1 - 169) in E. coli
            with amino terminal 6xHIS and FLAG tags that can be removed with TEV
            protease..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4533)
  AUTHORS   Hunter JC, Manandhar A, Carrasco MA, Gurbani D, Gondi S, Westover KD
  TITLE     Biochemical and Structural Analysis of Common Cancer-Associated KRAS
            Mutations.
  JOURNAL   Mol Cancer Res. 2015 Sep;13(9):1325-35. doi: 
            10.1158/1541-7786.MCR-15-0203. Epub 2015 Jun 2.
  PUBMED    26037647
REFERENCE   2  (bases 1 to 4533)
  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"; doi: 
            "10.1158/1541-7786.MCR-15-0203"; journalName: "Mol Cancer Res"; 
            date: "2015-09"; volume: "13"; issue: "9"; pages: "1325-35"
COMMENT     Sequence Label: pDNA2.0 6H-FLAG-TEV-KRAS G12C
FEATURES             Location/Qualifiers
     source          1..4533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      82..670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     571..590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     824..841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     939..956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     940..956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1413..1429
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        join(1454..1468,1469..1474,1475..1491,1492..1498)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1474..1490
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    1504..1523
                     /label=lac operator (symmetric)
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             1582..1599
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1609..1632
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1645..1665
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2377..2396)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2463..2554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2555..3364
                     /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"
     CDS             3447..4529
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3466..3485)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt
       61 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
      121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
      241 gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      841 ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac
      901 ggatggcctt tttgcgtttc tacaaactct ttctgtgttg taaaacgacg gccagtctta
      961 agctcgggcc ccctgggcgg ttctgataac gagtaatcgt taatccgcaa ataacgtaaa
     1021 aacccgcttc ggcgggtttt tttatggggg gagtttaggg aaaaagcatt tgtcagaata
     1081 tttaagggcg cctgtcactt tgcttgatat atgagaatta tttaacctta taaatgagaa
     1141 aaaagcaacg cactttaaat aagatacgtt gctttttcga ttgatgaaca cctataatta
     1201 aactattcat ctattattta tgattttttg tatatacaat atttctagtt tgttaaagag
     1261 aattaagaaa ataaatctcg aaaataataa agggaaaatc agtttttgat atcaaaatta
     1321 tacatgtcaa cgataataca aaatataata caaactataa gatgttatca gtatttatta
     1381 tgcatttaga ataaattttg tgtcgccctt aattgtgagc ggataacaat tacgagcttc
     1441 atgcacagtg aaatcatgaa aaatttattt gctttgtgag cggataacaa ttataatatg
     1501 tggaattgtg agcgctcaca attccacaac ggtttccctc tagaaataat tttgtttaac
     1561 ttttaggaga taaaacatat gcaccaccat catcatcatg ggggagggga ttacaaggat
     1621 gacgatgaca agtcttccgg ccgtgaaaat ctgtacttcc aaggcatgac ggagtacaaa
     1681 ctggtggttg tcggtgcgtg cggtgttggc aaaagcgcgc tgacgattca attgatccaa
     1741 aaccactttg tcgacgaata cgacccgacc attgaggatt cgtaccgtaa gcaggttgtc
     1801 atcgatggtg agacttgtct gttggacatc ctggacaccg caggccaaga agagtatagc
     1861 gccatgcgtg atcagtacat gcgtaccggt gagggctttc tgtgcgtgtt cgcgatcaac
     1921 aataccaaaa gcttcgagga cattcaccac tatcgtgagc aaatcaagcg cgttaaagat
     1981 agcgaagatg tgccgatggt gctggttggt aacaaatgtg atctgccaag ccgcaccgtg
     2041 gacaccaagc aggcccagga tctggcgcgt agctatggta ttccgtttat tgaaacgagc
     2101 gctaaaaccc gtcagggtgt cgacgacgca ttctatacgc tggtacgcga aatccgcaag
     2161 cacaaggaga agtaactcga gccccaaggg cgacaccccc taattagccc gggcgaaagg
     2221 cccagtcttt cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgca
     2281 tggggagtcc ccacactacc atcggcgcta cggcgtttca cttctgagtt cggcatgggg
     2341 tcaggtggga ccaccgcgct actgccgcca ggcaaacaag gggtgttatg agccatattc
     2401 aggtataaat gggctcgcga taatgttcag aattggttaa ttggttgtaa cactgacccc
     2461 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2521 ataaatgctt caataatatt gaaaaaggaa gaatatgagc catattcaac gggaaacgtc
     2581 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     2641 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     2701 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     2761 actaaactgg ctgacggaat ttatgccact tccgaccatc aagcatttta tccgtactcc
     2821 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcgttcc aggtattaga
     2881 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     2941 gcactcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gcctcgctca
     3001 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     3061 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     3121 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     3181 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     3241 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     3301 tggtattgat aatcctgata tgaataaatt gcaatttcat ttgatgctcg atgagttttt
     3361 ctaagcggcg cgccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     3421 aagagagtca attcagggtg gtgaatatga aaccagtaac gttatacgat gtcgcagagt
     3481 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     3541 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     3601 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     3661 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     3721 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     3781 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     3841 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     3901 accagacacc catcaacagt attattttct cccatgagga cggtacgcga ctgggcgtgg
     3961 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     4021 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     4081 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     4141 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     4201 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     4261 gatacgacga taccgaagat agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     4321 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     4381 cggtgaaggg caatcagctg ttgccagtct cactggtgaa aagaaaaacc accctggcgc
     4441 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4501 aggtttcccg actggaaagc gggcagtgac tca
//
