LOCUS       sequence-313312-        7404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Avi-Hs.KRAS4b(2-188) 
            G13D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7404)
  AUTHORS   Gillette WK, Esposito D, Abreu Blanco M, Alexander P, Bindu L, 
            Bittner C, Chertov O, Frank PH, Grose C, Jones JE, Meng Z, Perkins 
            S, Van Q, Ghirlando R, Fivash M, Nissley DV, McCormick F, 
            Holderfield M, Stephen AG
  TITLE     Farnesylated and methylated KRAS4b: high yield production of protein
            suitable for biophysical studies of prenylated protein-lipid 
            interactions.
  JOURNAL   Sci Rep. 2015 Nov 2;5:15916. doi: 10.1038/srep15916.
  PUBMED    26522388
REFERENCE   2  (bases 1 to 7404)
  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: "Sci Rep."; 
            date: "2015-11-2"; volume: "5:15916. doi"; pages: " 
            10.1038/srep15916"
COMMENT     Sequence Label: R701-X22-566: His6-MBP-tev-Avi-Hs.KRAS4b(2-188...
FEATURES             Location/Qualifiers
     source          1..7404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             120..1217
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1074..1083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1191..1214
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1236..1260
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1260..1280
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1278..1322
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1326..1889
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="TEYKLVVVGAGDVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVI
                     DGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKD
                     SEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIR
                     KHKEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(1888..1912)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1972..2004
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             2011..2028
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2194..2280
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2372..2399
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2453..2471)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2467..2514
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2551..3006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2638..2657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2848..2869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3121..3912,3913..3981))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3744..3763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3982..4074)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4155..4743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4644..4663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4897..4914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4929..5068
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4994..5013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5154..5176
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5170..5361)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5933..7015)
                     /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     6977..6996
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7016..7093)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7299..7318
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatccca tcaccatcac catcacggca
      121 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      181 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      241 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
      301 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
      361 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
      421 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
      481 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
      541 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
      601 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
      661 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
      721 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
      781 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
      841 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
      901 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
      961 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1021 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacca
     1081 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1141 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1201 tgaaagacgc gcagactaat tcgggatctg atatcacaag tttgtacaaa aaagttggcg
     1261 aaaacctgta cttccaaggc ctgaacgaca tcttcgaggc tcagaaaatc gaatggcacg
     1321 aaggcactga atataaactt gtggtagttg gagctggtga cgtaggcaag agtgccttga
     1381 cgatacagct aattcagaat cattttgtgg acgaatatga tccaacaata gaggattcct
     1441 acaggaagca agtagtaatt gatggagaaa cctgtctctt ggatattctc gacacagcag
     1501 gtcaagagga gtacagtgca atgagggacc agtacatgag gactggggag ggctttcttt
     1561 gtgtatttgc cataaataat actaaatcat ttgaagatat tcaccattat agagaacaaa
     1621 ttaaaagagt taaggactct gaagatgtac ctatggtcct agtaggaaat aaatgtgatt
     1681 tgccttctag aacagtagac acaaaacagg ctcaggactt agcaagaagt tatggaattc
     1741 cttttattga aacatcagca aagacaagac agggtgttga tgatgccttc tatacattag
     1801 ttcgagaaat tcgaaaacat aaagaaaaga tgagcaaaga tggtaaaaag aagaaaaaga
     1861 agtcaaagac aaagtgtgta attatgtaat caactttctt gtacaaagtg gtgatatcgc
     1921 tagctagata gctagaagct tgcggccgca cagctgtata cacgtgcaag ccagccagaa
     1981 ctcgctcctg aagacccaga ggatctcgag caccaccacc accaccacta atgttaatta
     2041 agttgggcgt tcctaggctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     2101 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     2161 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     2221 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     2281 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     2341 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     2401 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctgag
     2461 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     2521 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     2581 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2641 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2701 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2761 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2821 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2881 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2941 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3001 caatttctgg cggcacgatg gcatgagatt atcaaaaagg atcttcacct agatcctttt
     3061 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3121 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3181 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3241 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3301 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3361 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3421 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3481 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3541 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3601 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3661 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3721 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3781 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3841 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3901 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3961 acggaaatgt tgaatactca tactcttcct ttttcaatca tgattgaagc atttatcagg
     4021 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggtc
     4081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4261 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4861 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4921 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     4981 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     5041 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     5101 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5161 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     5221 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     5281 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     5341 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     5401 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     5461 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     5521 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     5581 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5641 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5701 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5761 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct
     5821 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5881 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5941 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6001 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     6061 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     6121 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     6181 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     6241 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     6301 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     6361 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     6421 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     6481 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     6541 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6601 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     6661 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     6721 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     6781 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     6841 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6901 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6961 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     7021 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     7081 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     7141 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     7201 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     7261 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     7321 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     7381 gagatcgatc tcgatcccgc gaaa
//
