LOCUS       sequence-313280-        7362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli 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 7362)
  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 7362)
  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-X28-566: His6-MBP-tev-G-Hs.KRAS4b(1-188) ...
FEATURES             Location/Qualifiers
     source          1..7362
                     /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             join(1281..1313,1314..1316,1317..1847)
                     /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             1281..1847
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(1846..1870)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1930..1962
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1969..1986
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2152..2238
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2330..2357
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2411..2429)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2425..2472
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2509..2964
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2596..2615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2806..2827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3079..3870,3871..3939))
                     /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     3702..3721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3940..4032)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4113..4701
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4602..4621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4855..4872
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4887..5026
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4952..4971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5112..5134
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5128..5319)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5891..6973)
                     /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     6935..6954
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6974..7051)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7257..7276
                     /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 atgactgaat ataaacttgt ggtagttgga gctgatggcg
     1321 taggcaagag tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc
     1381 caacaataga ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg
     1441 atattctcga cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga
     1501 ctggggaggg ctttctttgt gtatttgcca taaataatac taaatcattt gaagatattc
     1561 accattatag agaacaaatt aaaagagtta aggactctga agatgtacct atggtcctag
     1621 taggaaataa atgtgatttg ccttctagaa cagtagacac aaaacaggct caggacttag
     1681 caagaagtta tggaattcct tttattgaaa catcagcaaa gacaagacag ggtgttgatg
     1741 atgccttcta tacattagtt cgagaaattc gaaaacataa agaaaagatg agcaaagatg
     1801 gtaaaaagaa gaaaaagaag tcaaagacaa agtgtgtaat tatgtaatca actttcttgt
     1861 acaaagtggt gatatcgcta gctagatagc tagaagcttg cggccgcaca gctgtataca
     1921 cgtgcaagcc agccagaact cgctcctgaa gacccagagg atctcgagca ccaccaccac
     1981 caccactaat gttaattaag ttgggcgttc ctaggctgat aaaacagaat ttgcctggcg
     2041 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     2101 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     2161 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     2221 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     2281 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     2341 ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc
     2401 aaatatgtat ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2461 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2521 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2581 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2641 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2701 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2761 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2821 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2881 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2941 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     3001 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3061 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3121 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3181 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3241 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3301 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3361 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3421 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3481 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3541 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3601 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3661 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3721 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3781 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3841 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3901 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     3961 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4021 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4081 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4141 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4201 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4261 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4321 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4381 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4441 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4501 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4561 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4621 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4681 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4741 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4801 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4861 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4921 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4981 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5041 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5101 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5161 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5221 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5281 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5341 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5401 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5461 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5521 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5581 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5641 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     5701 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     5761 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5821 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5881 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5941 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     6001 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     6061 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     6121 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     6181 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     6241 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     6301 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     6361 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     6421 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     6481 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     6541 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     6601 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     6661 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6721 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6781 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6841 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6901 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6961 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     7021 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     7081 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     7141 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     7201 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     7261 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     7321 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aa
//
