LOCUS       sequence-493232-        5854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His6 tag-Avi tag-TEV cleavage site-KRAS (residues 1-174) 
            in E. coli, under the T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5854)
  AUTHORS   Wallon L, Khan I, Teng KW, Koide A, Zuberi M, Li J, Ketavarapu G, 
            Traaseth NJ, O'Bryan JP, Koide S
  TITLE     Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS 
            monobody targeting the Switch I/II pocket.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Oct 25;119(43):e2204481119. doi: 
            10.1073/pnas.2204481119. Epub 2022 Oct 17.
  PUBMED    36252024
REFERENCE   2  (bases 1 to 5854)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-10-25"; volume: "119(43):e2204481119. 
            doi"; pages: " 10.1073/pnas.2204481119. Epub 2022 Oct 17"
COMMENT     Sequence Label: pHBT-KRAS174
FEATURES             Location/Qualifiers
     source          1..5854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..23
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    23..47
                     /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)."
     RBS             62..84
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             122..166
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             167..187
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             722..739
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(792..810)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      806..853
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      890..1345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(977..996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1187..1208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1437..2252)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1437..2252)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2160..2179
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2244..2263
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2374..2962
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2863..2882
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3116..3133
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3148..3287
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3213..3232)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3373..3395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3389..3580)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4389..5471)
                     /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     5433..5452
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5472..5549)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5755..5774
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
       61 ttttgtttaa ctttaagaag gagatataca tatgaaacat catcatcatc atcacagcag
      121 cggcctgaat gatatttttg aagcacaaaa aattgaatgg catgaagaaa acctgtactt
      181 ccagggatcc atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag
      241 tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga
      301 ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga
      361 cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg
      421 ctttctttgt gtatttgcca taaataatac taaatcattt gaagatattc accattatag
      481 agaacaaatt aaaagagtta aggactctga ggatgtacct atggtcctag taggaaataa
      541 atgtgatttg ccttccagaa cagtagacac aaaacaggct caggacttag caagaagtta
      601 tggaattcct tttattgaaa catcagcaaa gacaagacag ggtgttgatg atgccttcta
      661 tacattagtt cgagaaattc gaaaacataa agaaaagatg agcaaagatg gttgactcga
      721 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
      781 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      841 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
      901 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      961 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1021 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1081 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1141 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1201 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1261 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1321 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1381 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     1441 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1501 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1561 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1621 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1681 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1741 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1801 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1861 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1921 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1981 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2041 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2101 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     2161 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     2221 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2281 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2341 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2401 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2461 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2521 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2581 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2641 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2701 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2761 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2821 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2881 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2941 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3001 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3061 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3121 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3181 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3241 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3301 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3361 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3421 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3481 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3541 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3601 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3661 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     3721 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     3781 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     3841 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     3901 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     3961 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4021 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4081 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4141 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4201 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4261 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4321 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4381 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4441 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4501 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4561 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4621 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4681 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4741 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4801 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     4861 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     4921 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     4981 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5041 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5101 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5161 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5221 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5281 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5341 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5401 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5461 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5521 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5581 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5641 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5701 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5761 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     5821 tccggcgtag aggatcgaga tctcgatccc gcga
//
