LOCUS       sequence-311636-        4500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KRAS G12D in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4500)
  AUTHORS   Poulin EJ, Bera AK, Lu J, Lin YJ, Strasser SD, Paulo JA, Huang TQ, 
            Morales C, Yan W, Cook J, Nowak JA, Brubaker DK, Joughin BA, Johnson
            CW, DeStefanis RA, Ghazi PC, Gondi S, Wales TE, Iacob RE, Bogdanova 
            L, Gierut JJ, Li Y, Engen JR, Perez-Mancera PA, Braun BS, Gygi SP, 
            Lauffenburger DA, Westover KD, Haigis KM
  TITLE     Tissue-Specific Oncogenic Activity of KRAS(A146T).
  JOURNAL   Cancer Discov. 2019 Jun;9(6):738-755. doi: 
            10.1158/2159-8290.CD-18-1220. Epub 2019 Apr 5.
  PUBMED    30952657
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: 
            "10.1158/2159-8290.CD-18-1220"; journalName: "Cancer Discov"; date: 
            "2019-06"; volume: "9"; issue: "6"; pages: "738-755"
COMMENT     Sequence Label: pDNA2.0-6H-TEV-KRAS G12D
FEATURES             Location/Qualifiers
     source          1..4500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..46
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     144..161
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     145..161
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    618..634
                     /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(659..673,674..679,680..696,697..703)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    679..695
                     /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    709..728
                     /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             787..804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             817..837
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1549..1568)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        1635..1726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1727..2536
                     /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             2619..3701
                     /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(2638..2657)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      3787..4375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4276..4295
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga
       61 gagtagggaa ctgccaggca tcaaactaag cagaaggccc ctgacggatg gcctttttgc
      121 gtttctacaa actctttctg tgttgtaaaa cgacggccag tcttaagctc gggccccctg
      181 ggcggttctg ataacgagta atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg
      241 gtttttttat ggggggagtt tagggaaaga gcatttgtca gaatatttaa gggcgcctgt
      301 cactttgctt gatatatgag aattatttaa ccttataaat gagaaaaaag caacgcactt
      361 taaataagat acgttgcttt ttcgattgat gaacacctat aattaaacta ttcatctatt
      421 atttatgatt ttttgtatat acaatatttc tagtttgtta aagagaatta agaaaataaa
      481 tctcgaaaat aataaaggga aaatcagttt ttgatatcaa aattatacat gtcaacgata
      541 atacaaaata taatacaaac tataagatgt tatcagtatt tattatgcat ttagaataaa
      601 ttttgtgtcg cccttaattg tgagcggata acaattacga gcttcatgca cagtgaaatc
      661 atgaaaaatt tatttgcttt gtgagcggat aacaattata atatgtggaa ttgtgagcgc
      721 tcacaattcc acaacggttt ccctctagaa ataattttgt ttaactttta ggagataaaa
      781 catatgcacc accatcatca tcattcttcc ggccgtgaaa atctgtactt ccaaggcatg
      841 acggagtaca aactggtggt tgtcggtgcg gacggtgttg gcaaaagcgc gctgacgatt
      901 caattgatcc aaaaccactt tgtcgacgaa tacgacccga ccattgagga ttcgtaccgt
      961 aagcaggttg tcatcgatgg tgagacttgt ctgttggaca tcctggacac cgcaggccaa
     1021 gaagagtata gcgccatgcg tgatcagtac atgcgtaccg gtgagggctt tctgtgcgtg
     1081 ttcgcgatca acaataccaa aagcttcgag gacattcacc actatcgtga gcaaatcaag
     1141 cgcgttaaag atagcgaaga tgtgccgatg gtgctggttg gtaacaaatg tgatctgcca
     1201 agccgcaccg tggacaccaa gcaggcccag gatctggcgc gtagctatgg tattccgttt
     1261 attgaaacga gcgctaaaac ccgtcagggt gtcgacgacg cattctatac gctggtacgc
     1321 gaaatccgca agcacaagga gaagtaactc gagccccaag ggcgacaccc cctaattagc
     1381 ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc
     1441 cctactctcg catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag
     1501 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta
     1561 tgagccatat tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt
     1621 aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     1681 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga gccatattca
     1741 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     1801 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     1861 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     1921 tgagatggtc agactaaact ggctgacgga atttatgcca cttccgacca tcaagcattt
     1981 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcgtt
     2041 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     2101 cctgcgccgg ttgcactcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     2161 tcgcctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     2221 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     2281 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     2341 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     2401 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     2461 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcaatttc atttgatgct
     2521 cgatgagttt ttctaagcgg cgcgccatcg aatggcgcaa aacctttcgc ggtatggcat
     2581 gatagcgccc ggaagagagt caattcaggg tggtgaatat gaaaccagta acgttatacg
     2641 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     2701 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     2761 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     2821 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     2881 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     2941 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     3001 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     3061 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgag gacggtacgc
     3121 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     3181 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     3241 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     3301 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     3361 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     3421 tctcggtagt gggatacgac gataccgaag atagctcatg ttatatcccg ccgttaacca
     3481 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     3541 ctcagggcca ggcggtgaag ggcaatcagc tgttgccagt ctcactggtg aaaagaaaaa
     3601 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3661 agctggcacg acaggtttcc cgactggaaa gcgggcagtg actcatgacc aaaatccctt
     3721 aacgtgagtt acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3781 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3841 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3901 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca
     3961 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4021 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4081 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4141 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4201 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4261 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4321 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4381 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4441 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
//
