LOCUS       sequence-313192-        6619 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculovirus protein expression of His6-MBP-tev-G-Hs.KRAS4b(1-188) 
            G12D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6619)
  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 6619)
  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: R989-X28-636: His6-MBP-tev-G-Hs.KRAS4b(1-188) ...
FEATURES             Location/Qualifiers
     source          1..6619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     17..34
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             146..163
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             167..1264
                     /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      1121..1130
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1238..1261
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1283..1307
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1307..1327
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1328..1360,1361..1363,1364..1894)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             1328..1894
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(1893..1917)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2062..2196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2099..2118)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2153..2172
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  2225..2390
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2574..3028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2661..2680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2870..2891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3054..3158
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3159..3227,3228..4019)
                     /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     complement(3377..3396)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4190..4778
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4679..4698
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4932..4949
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  5083..5307
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(5374..5907)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(6096..6101,6102..6118,6119..6124))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        complement(6487..6596)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
ORIGIN
        1 atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc
       61 gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca
      121 tcgggcgcgg atcaccatgg gatcccatca ccatcaccat cacggcaaaa tcgaagaagg
      181 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
      241 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
      301 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
      361 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
      421 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
      481 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
      541 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
      601 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
      661 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
      721 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
      781 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
      841 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
      901 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
      961 tattaacgct gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     1021 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     1081 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccaccatgg aaaacgccca
     1141 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     1201 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     1261 gactaattcg ggatcctcga tcacaagttt gtacaaaaaa gttggcgaaa acctgtactt
     1321 ccaaggcatg actgaatata aacttgtggt agttggagct gatggcgtag gcaagagtgc
     1381 cttgacgata cagctaattc agaatcattt tgtggacgaa tatgatccaa caatagagga
     1441 ttcctacagg aagcaagtag taattgatgg agaaacctgt ctcttggata ttctcgacac
     1501 agcaggtcaa gaggagtaca gtgcaatgag ggaccagtac atgaggactg gggagggctt
     1561 tctttgtgta tttgccataa ataatactaa atcatttgaa gatattcacc attatagaga
     1621 acaaattaaa agagttaagg actctgaaga tgtacctatg gtcctagtag gaaataaatg
     1681 tgatttgcct tctagaacag tagacacaaa acaggctcag gacttagcaa gaagttatgg
     1741 aattcctttt attgaaacat cagcaaagac aagacagggt gttgatgatg ccttctatac
     1801 attagttcga gaaattcgaa aacataaaga aaagatgagc aaagatggta aaaagaagaa
     1861 aaagaagtca aagacaaagt gtgtaattat gtaatcaact ttcttgtaca aagtggtgat
     1921 cgagctagct agaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt
     1981 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2041 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2101 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2161 tccaaactca tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctaggaga
     2221 tccgaaccag ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt
     2281 agcttacgac gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta
     2341 aaaactccat ttccacccct cccagttccc aactattttg tccgcccaca gcggggcatt
     2401 tttcttcctg ttatgttttt aatcaaacat cctgccaact ccatgtgaca aaccgtcatc
     2461 ttcggctact ttttctctgt cacagaatga aaatttttct gtcatctctt cgttattaat
     2521 gtttgtaatt gactgaatat caacgcttat ttgcagcctg aatggcgaat gggacgcgcc
     2581 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2641 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2701 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2761 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2821 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttcttaatag tggactcttg
     2881 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2941 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3001 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     3061 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3121 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3181 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3241 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3301 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3361 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3421 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3481 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3541 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3601 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3661 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3721 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3781 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3841 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3901 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3961 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4021 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4081 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4141 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4201 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4261 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4321 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4381 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4441 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4501 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4561 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4621 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4681 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4741 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4801 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4861 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4921 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4981 tccttacgca tctgtgcggt atttcacacc gcatagacca gccgcgtaac ctggcaaaat
     5041 cggttacggt tgagtaataa atggatgccc tgcgtaagcg ggtgtgggcg gacaataaag
     5101 tcttaaactg aacaaaatag atctaaacta tgacaataaa gtcttaaact agacagaata
     5161 gttgtaaact gaaatcagtc cagttatgct gtgaaaaagc atactggact tttgttatgg
     5221 ctaaagcaaa ctcttcattt tctgaagtgc aaattgcccg tcgtattaaa gaggggcgtg
     5281 gccaagggca tggtaaagac tatattcgcg gcgttgtgac aatttaccga acaactccgc
     5341 ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg gcggtacttg ggtcgatatc
     5401 aaagtgcatc acttcttccc gtatgcccaa ctttgtatag agagccactg cgggatcgtc
     5461 accgtaatct gcttgcacgt agatcacata agcaccaagc gcgttggcct catgcttgag
     5521 gagattgatg agcgcggtgg caatgccctg cctccggtgc tcgccggaga ctgcgagatc
     5581 atagatatag atctcactac gcggctgctc aaacttgggc agaacgtaag ccgcgagagc
     5641 gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg aatgtcttac tacggagcaa
     5701 gttcccgagg taatcggagt ccggctgatg ttgggagtag gtggctacgt ctccgaactc
     5761 acgaccgaaa agatcaagag cagcccgcat ggatttgact tggtcagggc cgagcctaca
     5821 tgtgcgaatg atgcccatac ttgagccacc taactttgtt ttagggcgac tgccctgctg
     5881 cgtaacatcg ttgctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca
     5941 cggcgtaacg cgcttgctgc ttggatgccc gaggcataga ctgtacaaaa aaacagtcat
     6001 aacaagccat gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg
     6061 accagttgcg tgagcgcata cgctacttgc attacagttt acgaaccgaa caggcttatg
     6121 tcaactgggt tcgtgccttc atccgtttcc acggtgtgcg tcacccggca accttgggca
     6181 gcagcgaagt cgaggcattt ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca
     6241 cgcatcgtca ggcattggcg gccttgctgt tcttctacgg caaggtgctg tgcacggatc
     6301 tgcccttgct tcaggagatc ggtagacctc ggccgtcgcg gcgcttgccg gtggtgctga
     6361 ccccggatga agtggttcgc atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc
     6421 aggactctag ctatagttct agtggttggc tactcgagat cccgggtgat caagtcttcg
     6481 tcgagtgatt gtaaataaaa tgtaatttac agtatagtat tttaattaat atacaaatga
     6541 tttgataata attcttattt aactataata tattgtgttg ggttgaatta aaggtccgta
     6601 tactccggaa tattaatag
//
