LOCUS       sequence-313281-        7347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Avi-Hs.KRAS4b(2-169) 
            G12D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7347)
  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 7347)
  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-X54-566: His6-MBP-tev-Avi-Hs.KRAS4b(2-169...
FEATURES             Location/Qualifiers
     source          1..7347
                     /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"
     protein_bind    complement(1831..1855)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1915..1947
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1954..1971
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2137..2223
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2315..2342
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2396..2414)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2410..2457
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2494..2949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2581..2600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2791..2812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3064..3855,3856..3924))
                     /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     3687..3706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3925..4017)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4098..4686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4587..4606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4840..4857
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4872..5011
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4937..4956)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5097..5119
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5113..5304)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5876..6958)
                     /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     6920..6939
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6959..7036)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7242..7261
                     /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 gagctgatgg 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 aaagaaaagt aatcaacttt cttgtacaaa gtggtgatat
     1861 cgctagctag atagctagaa gcttgcggcc gcacagctgt atacacgtgc aagccagcca
     1921 gaactcgctc ctgaagaccc agaggatctc gagcaccacc accaccacca ctaatgttaa
     1981 ttaagttggg cgttcctagg ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     2041 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     2101 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     2161 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     2221 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     2281 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     2341 tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct
     2401 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2461 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2521 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2581 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2641 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2701 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2761 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2821 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2881 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     2941 ttacaatttc tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct
     3001 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3061 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3121 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3181 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3241 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3301 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3361 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3421 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3481 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3541 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3601 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3661 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3721 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3781 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3841 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3901 gacacggaaa tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc
     3961 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4021 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4081 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4141 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4201 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4261 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4321 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4381 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4441 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4501 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4561 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4621 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4681 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4741 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4801 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4861 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4921 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4981 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5041 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5101 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5161 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5221 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5281 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5341 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5401 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5461 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5521 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5581 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5641 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5701 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5761 gctagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5821 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5881 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5941 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     6001 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     6061 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     6121 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     6181 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     6241 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6301 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6361 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6421 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6481 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6541 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6601 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6661 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6721 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6781 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6841 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6901 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6961 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     7021 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     7081 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     7141 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     7201 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     7261 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     7321 atcgagatcg atctcgatcc cgcgaaa
//
