LOCUS       sequence-311637-        4533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human KRAS P34R 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 4533)
  AUTHORS   Bera AK, Lu J, Lu C, Li L, Gondi S, Yan W, Nelson A, Zhang G, 
            Westover KD
  TITLE     GTP hydrolysis is modulated by Arg34 in the RASopathy-associated 
            KRAS(P34R).
  JOURNAL   Birth Defects Res. 2020 Mar 18. doi: 10.1002/bdr2.1647.
  PUBMED    32187889
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"; journalName: "Birth 
            Defects Res. 2020 Mar 18. doi: 10.1002/bdr2.1647."
COMMENT     Sequence Label: KRAS-P34R
FEATURES             Location/Qualifiers
     source          1..4533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..107
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     91..107
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    564..580
                     /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(605..619,620..625,626..642,643..649)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    625..641
                     /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    655..674
                     /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             733..750
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             760..783
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             796..816
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1528..1547)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        1614..1705
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1706..2515
                     /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             2598..3680
                     /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(2617..2636)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      3766..4354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4255..4274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4508..4525
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga cggatggcct
       61 ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt aagctcgggc
      121 cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa aaacccgctt
      181 cggcgggttt ttttatgggg ggagtttagg gaaaaagcat ttgtcagaat atttaagggc
      241 gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga aaaaagcaac
      301 gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt aaactattca
      361 tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga gaattaagaa
      421 aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt atacatgtca
      481 acgataatac aaaatataat acaaactata agatgttatc agtatttatt atgcatttag
      541 aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt
      601 gaaatcatga aaaatttatt tgctttgtga gcggataaca attataatat gtggaattgt
      661 gagcgctcac aattccacaa cggtttccct ctagaaataa ttttgtttaa cttttaggag
      721 ataaaacata tgcaccacca tcatcatcat gggggagggg attacaagga tgacgatgac
      781 aagtcttccg gccgtgaaaa tctgtacttc caaggcatga cggagtacaa actggtggtt
      841 gtcggtgcgg gcggtgttgg caaaagcgcg ctgacgattc aattgatcca aaaccacttt
      901 gtcgacgaat acgaccgcac cattgaggat tcgtaccgta agcaggttgt catcgatggt
      961 gagacttgtc tgttggacat cctggacacc gcaggccaag aagagtatag cgccatgcgt
     1021 gatcagtaca tgcgtaccgg tgagggcttt ctgtgcgtgt tcgcgatcaa caataccaaa
     1081 agcttcgagg acattcacca ctatcgtgag caaatcaagc gcgttaaaga tagcgaagat
     1141 gtgccgatgg tgctggttgg taacaaatgt gatctgccaa gccgcaccgt ggacaccaag
     1201 caggcccagg atctggcgcg tagctatggt attccgttta ttgaaacgag cgctaaaacc
     1261 cgtcagggtg tcgacgacgc attctatacg ctggtacgcg aaatccgcaa gcacaaggag
     1321 aagtaactcg agccccaagg gcgacacccc ctaattagcc cgggcgaaag gcccagtctt
     1381 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagtc
     1441 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
     1501 accaccgcgc tactgccgcc aggcaaacaa ggggtgttat gagccatatt caggtataaa
     1561 tgggctcgcg ataatgttca gaattggtta attggttgta acactgaccc ctatttgttt
     1621 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1681 tcaataatat tgaaaaagga agaatatgag ccatattcaa cgggaaacgt cgaggccgcg
     1741 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     1801 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     1861 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     1921 gctgacggaa tttatgccac ttccgaccat caagcatttt atccgtactc ctgatgatgc
     1981 atggttactc accactgcga tccccggaaa aacagcgttc caggtattag aagaatatcc
     2041 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcactcgat
     2101 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc
     2161 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     2221 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     2281 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     2341 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     2401 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     2461 taatcctgat atgaataaat tgcaatttca tttgatgctc gatgagtttt tctaagcggc
     2521 gcgccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     2581 aattcagggt ggtgaatatg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     2641 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     2701 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     2761 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     2821 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     2881 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     2941 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     3001 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     3061 ccatcaacag tattattttc tcccatgagg acggtacgcg actgggcgtg gagcatctgg
     3121 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     3181 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     3241 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     3301 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     3361 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     3421 ataccgaaga tagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     3481 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     3541 gcaatcagct gttgccagtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     3601 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3661 gactggaaag cgggcagtga ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc
     3721 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3781 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3841 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3901 accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga actctgtagc
     3961 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4021 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4081 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4141 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4201 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4261 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4321 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4381 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4441 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4501 cgagcgcagc gagtcagtga gcgaggaagc gga
//
