LOCUS       sequence-308189-        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"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1417..1433
                     /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(1458..1472,1473..1478,1479..1495,1496..1502)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1478..1494
                     /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    1508..1527
                     /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             1586..1603
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1616..1636
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2348..2367)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2434..2525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2526..3335
                     /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             3418..4500
                     /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(3437..3456)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatgcatt tagaataaat tttgtgtcgc ccttaattgt gagcggataa caattacgag
     1441 cttcatgcac agtgaaatca tgaaaaattt atttgctttg tgagcggata acaattataa
     1501 tatgtggaat tgtgagcgct cacaattcca caacggtttc cctctagaaa taattttgtt
     1561 taacttttag gagataaaac atatgcacca ccatcatcat cattcttccg gccgtgaaaa
     1621 tctgtacttc caaggcatga cggagtacaa actggtggtt gtcggtgcgg acggtgttgg
     1681 caaaagcgcg ctgacgattc aattgatcca aaaccacttt gtcgacgaat acgacccgac
     1741 cattgaggat tcgtaccgta agcaggttgt catcgatggt gagacttgtc tgttggacat
     1801 cctggacacc gcaggccaag aagagtatag cgccatgcgt gatcagtaca tgcgtaccgg
     1861 tgagggcttt ctgtgcgtgt tcgcgatcaa caataccaaa agcttcgagg acattcacca
     1921 ctatcgtgag caaatcaagc gcgttaaaga tagcgaagat gtgccgatgg tgctggttgg
     1981 taacaaatgt gatctgccaa gccgcaccgt ggacaccaag caggcccagg atctggcgcg
     2041 tagctatggt attccgttta ttgaaacgag cgctaaaacc cgtcagggtg tcgacgacgc
     2101 attctatacg ctggtacgcg aaatccgcaa gcacaaggag aagtaactcg agccccaagg
     2161 gcgacacccc ctaattagcc cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt
     2221 atttgatgcc tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct
     2281 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     2341 aggcaaacaa ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca
     2401 gaattggtta attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa
     2461 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2521 agaatatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg
     2581 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     2641 atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     2701 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgccac
     2761 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     2821 tccccggaaa aacagcgttc caggtattag aagaatatcc tgattcaggt gaaaatattg
     2881 ttgatgcgct ggcagtgttc ctgcgccggt tgcactcgat tcctgtttgt aattgtcctt
     2941 ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc acgaatgaat aacggtttgg
     3001 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     3061 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     3121 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     3181 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     3241 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     3301 tgcagtttca tttgatgctc gatgagtttt tctaagcggc gcgccatcga atggcgcaaa
     3361 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatatg
     3421 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     3481 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     3541 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     3601 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     3661 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     3721 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     3781 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     3841 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     3901 tcccatgagg acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     3961 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     4021 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     4081 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     4141 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     4201 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga tagctcatgt
     4261 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     4321 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgccagtc
     4381 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     4441 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
//
