LOCUS       sequence-213465-        4500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses activated human KRAS G13D (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 G13D
FEATURES             Location/Qualifiers
     source          1..4500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      78..666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     567..586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     820..837
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     935..952
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     936..952
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1409..1425
                     /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(1450..1464,1465..1470,1471..1487,1488..1494)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1470..1486
                     /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    1500..1519
                     /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             1578..1595
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1608..1628
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2340..2359)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2426..2517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2518..3327
                     /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             3410..4492
                     /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(3429..3448)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 caaaatccct taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga ccccgtagaa
       61 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      121 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      181 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      241 tagttagccc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      301 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      361 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      421 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      481 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      541 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      601 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      661 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      721 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
      781 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
      841 gcggaaggcg agagtaggga actgccaggc atcaaactaa gcagaaggcc cctgacggat
      901 ggcctttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct
      961 cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc
     1021 cgcttcggcg ggttttttta tggggggagt ttagggaaag agcatttgtc agaatattta
     1081 agggcgcctg tcactttgct tgatatatga gaattattta accttataaa tgagaaaaaa
     1141 gcaacgcact ttaaataaga tacgttgctt tttcgattga tgaacaccta taattaaact
     1201 attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt aaagagaatt
     1261 aagaaaataa atctcgaaaa taataaaggg aaaatcagtt tttgatatca aaattataca
     1321 tgtcaacgat aatacaaaat ataatacaaa ctataagatg ttatcagtat ttattatgca
     1381 tttagaataa attttgtgtc gcccttaatt gtgagcggat aacaattacg agcttcatgc
     1441 acagtgaaat catgaaaaat ttatttgctt tgtgagcgga taacaattat aatatgtgga
     1501 attgtgagcg ctcacaattc cacaacggtt tccctctaga aataattttg tttaactttt
     1561 aggagataaa acatatgcac caccatcatc atcattcttc cggccgtgaa aatctgtact
     1621 tccaaggcat gacggagtac aaactggtgg ttgtcggtgc gggcgatgtt ggcaaaagcg
     1681 cgctgacgat tcaattgatc caaaaccact ttgtcgacga atacgacccg accattgagg
     1741 attcgtaccg taagcaggtt gtcatcgatg gtgagacttg tctgttggac atcctggaca
     1801 ccgcaggcca agaagagtat agcgccatgc gtgatcagta catgcgtacc ggtgagggct
     1861 ttctgtgcgt gttcgcgatc aacaatacca aaagcttcga ggacattcac cactatcgtg
     1921 agcaaatcaa gcgcgttaaa gatagcgaag atgtgccgat ggtgctggtt ggtaacaaat
     1981 gtgatctgcc aagccgcacc gtggacacca agcaggccca ggatctggcg cgtagctatg
     2041 gtattccgtt tattgaaacg agcgctaaaa cccgtcaggg tgtcgacgac gcattctata
     2101 cgctggtacg cgaaatccgc aagcacaagg agaagtaact cgagccccaa gggcgacacc
     2161 ccctaattag cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     2221 cctggcagtt ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt
     2281 tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac
     2341 aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt
     2401 taattggttg taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2461 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg
     2521 agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta
     2581 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
     2641 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     2701 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc acttccgacc
     2761 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga
     2821 aaaacagcgt tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     2881 ctggcagtgt tcctgcgccg gttgcactcg attcctgttt gtaattgtcc ttttaacagc
     2941 gatcgcgtat ttcgcctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     3001 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     3061 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     3121 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     3181 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     3241 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcaattt
     3301 catttgatgc tcgatgagtt tttctaagcg gcgcgccatc gaatggcgca aaacctttcg
     3361 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaata tgaaaccagt
     3421 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     3481 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     3541 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     3601 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     3661 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     3721 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     3781 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     3841 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     3901 ggacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     3961 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     4021 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     4081 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     4141 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     4201 tggtgcggat atctcggtag tgggatacga cgataccgaa gatagctcat gttatatccc
     4261 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     4321 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgccag tctcactggt
     4381 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     4441 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gactcatgac
//
