LOCUS       sequence-311639-        4533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A146T Kras with Flag tag inserted into N-terminal domain between 
            6Xhis tag and TEV cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4533)
  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 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: 
            "10.1158/2159-8290.CD-18-1220"; journalName: "Cancer Discov"; date: 
            "2019-06"; volume: "9"; issue: "6"; pages: "738-755"
COMMENT     Sequence Label: KRAS-A146T
FEATURES             Location/Qualifiers
     source          1..4533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     95..112
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     96..112
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..585
                     /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(610..624,625..630,631..647,648..654)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    630..646
                     /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    660..679
                     /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             738..755
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             765..788
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             801..821
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1533..1552)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        1619..1710
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1711..2520
                     /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             2603..3685
                     /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(2622..2641)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      3771..4359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4260..4279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4513..4530
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcggaaggcg agagtaggga actgccaggc atcaaactaa gcagaaggcc cctgacggat
       61 ggcctttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct
      121 cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc
      181 cgcttcggcg ggttttttta tggggggagt ttagggaaaa agcatttgtc agaatattta
      241 agggcgcctg tcactttgct tgatatatga gaattattta accttataaa tgagaaaaaa
      301 gcaacgcact ttaaataaga tacgttgctt tttcgattga tgaacaccta taattaaact
      361 attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt aaagagaatt
      421 aagaaaataa atctcgaaaa taataaaggg aaaatcagtt tttgatatca aaattataca
      481 tgtcaacgat aatacaaaat ataatacaaa ctataagatg ttatcagtat ttattatgca
      541 tttagaataa attttgtgtc gcccttaatt gtgagcggat aacaattacg agcttcatgc
      601 acagtgaaat catgaaaaat ttatttgctt tgtgagcgga taacaattat aatatgtgga
      661 attgtgagcg ctcacaattc cacaacggtt tccctctaga aataattttg tttaactttt
      721 aggagataaa acatatgcac caccatcatc atcatggggg aggggattac aaggatgacg
      781 atgacaagtc ttccggccgt gaaaatctgt acttccaagg catgacggag tacaaactgg
      841 tggttgtcgg tgcgggcggt gttggcaaaa gcgcgctgac gattcaattg atccaaaacc
      901 actttgtcga cgaatacgac ccgaccattg aggattcgta ccgtaagcag gttgtcatcg
      961 atggtgagac ttgtctgttg gacatcctgg acaccgcagg ccaagaagag tatagcgcca
     1021 tgcgtgatca gtacatgcgt accggtgagg gctttctgtg cgtgttcgcg atcaacaata
     1081 ccaaaagctt cgaggacatt caccactatc gtgagcaaat caagcgcgtt aaagatagcg
     1141 aagatgtgcc gatggtgctg gttggtaaca aatgtgatct gccaagccgc accgtggaca
     1201 ccaagcaggc ccaggatctg gcgcgtagct atggtattcc gtttattgaa acgagcacca
     1261 aaacccgtca gggtgtcgac gacgcattct atacgctggt acgcgaaatc cgcaagcaca
     1321 aggagaagta actcgagccc caagggcgac accccctaat tagcccgggc gaaaggccca
     1381 gtctttcgac tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg
     1441 gagtccccac actaccatcg gcgctacggc gtttcacttc tgagttcggc atggggtcag
     1501 gtgggaccac cgcgctactg ccgccaggca aacaaggggt gttatgagcc atattcaggt
     1561 ataaatgggc tcgcgataat gttcagaatt ggttaattgg ttgtaacact gacccctatt
     1621 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1681 atgcttcaat aatattgaaa aaggaagaat atgagccata ttcaacggga aacgtcgagg
     1741 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     1801 gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg
     1861 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     1921 aactggctga cggaatttat gccacttccg accatcaagc attttatccg tactcctgat
     1981 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cgttccaggt attagaagaa
     2041 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcac
     2101 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgcct cgctcaggcg
     2161 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     2221 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     2281 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     2341 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     2401 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     2461 attgataatc ctgatatgaa taaattgcaa tttcatttga tgctcgatga gtttttctaa
     2521 gcggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     2581 gagtcaattc agggtggtga atatgaaacc agtaacgtta tacgatgtcg cagagtatgc
     2641 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     2701 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     2761 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     2821 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     2881 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     2941 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     3001 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     3061 gacacccatc aacagtatta ttttctccca tgaggacggt acgcgactgg gcgtggagca
     3121 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     3181 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     3241 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     3301 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     3361 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     3421 cgacgatacc gaagatagct catgttatat cccgccgtta accaccatca aacaggattt
     3481 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     3541 gaagggcaat cagctgttgc cagtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     3601 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     3661 ttcccgactg gaaagcgggc agtgactcat gaccaaaatc ccttaacgtg agttacgcgc
     3721 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3781 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3841 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3901 cagataccaa atactgttct tctagtgtag ccgtagttag cccaccactt caagaactct
     3961 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4021 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4081 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4141 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4201 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4261 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4321 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4381 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4441 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4501 acgaccgagc gcagcgagtc agtgagcgag gaa
//
