LOCUS       sequence-311638-        4533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  V14I 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   Bera AK, Lu J, Wales TE, Gondi S, Gurbani D, Nelson A, Engen JR, 
            Westover KD
  TITLE     Structural basis of the atypical activation mechanism of KRAS(V14I).
  JOURNAL   J Biol Chem. 2019 Sep 20;294(38):13964-13972. doi: 
            10.1074/jbc.RA119.009131. Epub 2019 Jul 24.
  PUBMED    31341022
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: 
            "10.1074/jbc.RA119.009131"; journalName: "J Biol Chem"; date: 
            "2019-09-20- 20"; volume: "294"; issue: "38"; pages: "13964-13972"
COMMENT     Sequence Label: KRAS-V14I
FEATURES             Location/Qualifiers
     source          1..4533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    244..260
                     /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(285..299,300..305,306..322,323..329)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    305..321
                     /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    335..354
                     /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             413..430
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             440..463
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             476..496
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1208..1227)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        1294..1385
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1386..2195
                     /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             2278..3360
                     /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(2297..2316)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      3446..4034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3935..3954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4188..4205
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4303..4320
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4304..4320
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgctttttcg attgatgaac acctataatt aaactattca tctattattt atgatttttt
       61 gtatatacaa tatttctagt ttgttaaaga gaattaagaa aataaatctc gaaaataata
      121 aagggaaaat cagtttttga tatcaaaatt atacatgtca acgataatac aaaatataat
      181 acaaactata agatgttatc agtatttatt atgcatttag aataaatttt gtgtcgccct
      241 taattgtgag cggataacaa ttacgagctt catgcacagt gaaatcatga aaaatttatt
      301 tgctttgtga gcggataaca attataatat gtggaattgt gagcgctcac aattccacaa
      361 cggtttccct ctagaaataa ttttgtttaa cttttaggag ataaaacata tgcaccacca
      421 tcatcatcat gggggagggg attacaagga tgacgatgac aagtcttccg gccgtgaaaa
      481 tctgtacttc caaggcatga cggagtacaa actggtggtt gtcggtgcgg gcggtattgg
      541 caaaagcgcg ctgacgattc aattgatcca aaaccacttt gtcgacgaat acgacccgac
      601 cattgaggat tcgtaccgta agcaggttgt catcgatggt gagacttgtc tgttggacat
      661 cctggacacc gcaggccaag aagagtatag cgccatgcgt gatcagtaca tgcgtaccgg
      721 tgagggcttt ctgtgcgtgt tcgcgatcaa caataccaaa agcttcgagg acattcacca
      781 ctatcgtgag caaatcaagc gcgttaaaga tagcgaagat gtgccgatgg tgctggttgg
      841 taacaaatgt gatctgccaa gccgcaccgt ggacaccaag caggcccagg atctggcgcg
      901 tagctatggt attccgttta ttgaaacgag cgctaaaacc cgtcagggtg tcgacgacgc
      961 attctatacg ctggtacgcg aaatccgcaa gcacaaggag aagtaactcg agccccaagg
     1021 gcgacacccc ctaattagcc cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt
     1081 atttgatgcc tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct
     1141 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     1201 aggcaaacaa ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca
     1261 gaattggtta attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa
     1321 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1381 agaatatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg
     1441 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     1501 atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     1561 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgccac
     1621 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     1681 tccccggaaa aacagcgttc caggtattag aagaatatcc tgattcaggt gaaaatattg
     1741 ttgatgcgct ggcagtgttc ctgcgccggt tgcactcgat tcctgtttgt aattgtcctt
     1801 ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc acgaatgaat aacggtttgg
     1861 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     1921 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     1981 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     2041 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     2101 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     2161 tgcaatttca tttgatgctc gatgagtttt tctaagcggc gcgccatcga atggcgcaaa
     2221 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatatg
     2281 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     2341 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     2401 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     2461 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     2521 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     2581 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     2641 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     2701 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     2761 tcccatgagg acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     2821 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     2881 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     2941 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     3001 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     3061 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga tagctcatgt
     3121 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     3181 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgccagtc
     3241 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     3301 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     3361 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
     3421 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3481 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3541 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3601 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3661 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3721 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3781 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3841 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3901 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3961 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4021 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4081 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4141 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4201 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
     4261 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
     4321 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     4381 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaaaag catttgtcag
     4441 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     4501 agaaaaaagc aacgcacttt aaataagata cgt
//
