LOCUS       sequence-493231-        5856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His6 tag-Avi tag-TEV cleavage site-HRAS (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 5856)
  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 5856)
  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-HRAS174
FEATURES             Location/Qualifiers
     source          1..5856
                     /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(794..812)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      808..855
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      892..1347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(979..998)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1189..1210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1439..2254)
                     /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(1439..2254)
                     /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     2162..2181
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2246..2265
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2376..2964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2865..2884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3118..3135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3150..3289
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3215..3234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3375..3397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3391..3582)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4391..5473)
                     /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     5435..5454
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5474..5551)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5757..5776
                     /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 atgacggaat ataagcttgt ggtggtgggc gccggcggtg tgggcaagag
      241 tgcgctgacc atccagctga tccagaacca ctttgtggac gaatacgacc ccactataga
      301 ggattcctac cggaagcagg tggtcattga tggggagacg tgcctgttgg acatcctgga
      361 taccgccggc caggaggagt acagcgccat gcgggaccag tacatgcgca ccggggaggg
      421 cttcctgtgt gtgtttgcca tcaacaacac caagtctttt gaggacatcc accagtacag
      481 ggagcagatc aaacgggtga aggactcgga tgacgtgccc atggtgctgg tggggaacaa
      541 gtgtgacctg gctgcacgca ctgtggaatc tcggcaggct caggacctcg cccgaagcta
      601 cggcatcccc tacatcgaga cctcggccaa gacccggcag ggagtggagg atgccttcta
      661 cacgttggtg cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc cttgactcga
      721 gcaccaccac caccaccact gagatccggc agcacaagct gcggaagctg aaccctcctt
      781 gactcgagca ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      841 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      901 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      961 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1021 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1081 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1141 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1201 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1261 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1321 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1381 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1441 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1501 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1561 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1621 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1681 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1741 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1801 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1861 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1921 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1981 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2041 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2101 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2161 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2221 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2281 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2341 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2401 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2461 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2521 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2581 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2641 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2701 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2761 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2821 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2881 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2941 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3001 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3061 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3121 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3181 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3241 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3301 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3361 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3421 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3481 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3541 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3601 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3661 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3721 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3781 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3841 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3901 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3961 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4021 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4081 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4141 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4201 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4261 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4321 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4381 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4441 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4501 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4561 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4621 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4681 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4741 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4801 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4861 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4921 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4981 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5041 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5101 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5161 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5221 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5281 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5341 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5401 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5461 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5521 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5581 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5641 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5701 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5761 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5821 cgtccggcgt agaggatcga gatctcgatc ccgcga
//
