LOCUS       sequence-213464-        4500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses activated human KRAS G12V (amino acids 1 - 169) in E. coli
            with amino terminal 6xHIS tag that can be removed with TEV 
            protease..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4500)
  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 4500)
  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-TEV-KRAS G12V
FEATURES             Location/Qualifiers
     source          1..4500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      83..671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     572..591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     825..842
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     940..957
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     941..957
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1414..1430
                     /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(1455..1469,1470..1475,1476..1492,1493..1499)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1475..1491
                     /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    1505..1524
                     /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             1583..1600
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1613..1633
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2345..2364)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2431..2522
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2523..3332
                     /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             3415..4497
                     /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(3434..3453)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atgaccaaaa tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg
       61 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
      121 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      181 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      241 agccgtagtt agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      301 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      361 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      421 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      481 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      541 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      601 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      661 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      721 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      781 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      841 aggaagcgga aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga
      901 cggatggcct ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt
      961 aagctcgggc cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa
     1021 aaacccgctt cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat
     1081 atttaagggc gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga
     1141 aaaaagcaac gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt
     1201 aaactattca tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga
     1261 gaattaagaa aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt
     1321 atacatgtca acgataatac aaaatataat acaaactata agatgttatc agtatttatt
     1381 atgcatttag aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt
     1441 catgcacagt gaaatcatga aaaatttatt tgctttgtga gcggataaca attataatat
     1501 gtggaattgt gagcgctcac aattccacaa cggtttccct ctagaaataa ttttgtttaa
     1561 cttttaggag ataaaacata tgcaccacca tcatcatcat tcttccggcc gtgaaaatct
     1621 gtacttccaa ggcatgacgg agtacaaact ggtggttgtc ggtgcggtcg gtgttggcaa
     1681 aagcgcgctg acgattcaat tgatccaaaa ccactttgtc gacgaatacg acccgaccat
     1741 tgaggattcg taccgtaagc aggttgtcat cgatggtgag acttgtctgt tggacatcct
     1801 ggacaccgca ggccaagaag agtatagcgc catgcgtgat cagtacatgc gtaccggtga
     1861 gggctttctg tgcgtgttcg cgatcaacaa taccaaaagc ttcgaggaca ttcaccacta
     1921 tcgtgagcaa atcaagcgcg ttaaagatag cgaagatgtg ccgatggtgc tggttggtaa
     1981 caaatgtgat ctgccaagcc gcaccgtgga caccaagcag gcccaggatc tggcgcgtag
     2041 ctatggtatt ccgtttattg aaacgagcgc taaaacccgt cagggtgtcg acgacgcatt
     2101 ctatacgctg gtacgcgaaa tccgcaagca caaggagaag taactcgagc cccaagggcg
     2161 acacccccta attagcccgg gcgaaaggcc cagtctttcg actgagcctt tcgttttatt
     2221 tgatgcctgg cagttcccta ctctcgcatg gggagtcccc acactaccat cggcgctacg
     2281 gcgtttcact tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg
     2341 caaacaaggg gtgttatgag ccatattcag gtataaatgg gctcgcgata atgttcagaa
     2401 ttggttaatt ggttgtaaca ctgaccccta tttgtttatt tttctaaata cattcaaata
     2461 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2521 atatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg
     2581 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     2641 gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     2701 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgccacttc
     2761 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     2821 ccggaaaaac agcgttccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     2881 atgcgctggc agtgttcctg cgccggttgc actcgattcc tgtttgtaat tgtcctttta
     2941 acagcgatcg cgtatttcgc ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     3001 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     3061 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     3121 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     3181 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     3241 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     3301 aatttcattt gatgctcgat gagtttttct aagcggcgcg ccatcgaatg gcgcaaaacc
     3361 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatatgaaa
     3421 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     3481 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     3541 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     3601 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     3661 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     3721 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     3781 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     3841 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     3901 catgaggacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     3961 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     4021 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     4081 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     4141 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     4201 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagatag ctcatgttat
     4261 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     4321 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gccagtctca
     4381 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     4441 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgactc
//
