LOCUS       sequence-313754-        7206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Hs.RAF1(52-188).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7206)
  AUTHORS   Lakshman B, Messing S, Schmid EM, Clogston JD, Gillette WK, Esposito
            D, Kessing B, Fletcher DA, Nissley DV, McCormick F, Stephen AG, 
            Jean-Francois FL
  TITLE     Quantitative biophysical analysis defines key components modulating 
            recruitment of the GTPase KRAS to the plasma membrane.
  JOURNAL   J Biol Chem. 2019 Feb 8;294(6):2193-2207. doi: 
            10.1074/jbc.RA118.005669. Epub 2018 Dec 17.
  PUBMED    30559287
REFERENCE   2  (bases 1 to 7206)
  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"; doi: 
            "10.1074/jbc.RA118.005669"; journalName: "J Biol Chem"; date: 
            "2019-02-8- 8"; volume: "294"; issue: "6"; pages: "2193-2207"
COMMENT     Sequence Label: R717-X57-566: His6-MBP-tev-Hs.RAF1(52-188)
FEATURES             Location/Qualifiers
     source          1..7206
                     /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="ENLYFQS"
     protein_bind    complement(1690..1714)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1774..1806
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1813..1830
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1996..2082
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2174..2201
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2255..2273)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2269..2316
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2353..2808
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2440..2459)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2650..2671
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2923..3714,3715..3783))
                     /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     3546..3565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3784..3876)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3957..4545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4446..4465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4699..4716
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4731..4870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4796..4815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4956..4978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4972..5163)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5735..6817)
                     /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     6779..6798
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6818..6895)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7101..7120
                     /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 cttccaatct aagacaagca acactatccg tgttttcttg ccgaacaagc
     1321 aaagaacagt ggtcaatgtg cgaaatggaa tgagcttgca tgactgcctt atgaaagcac
     1381 tcaaggtgag gggcctgcaa ccagagtgct gtgcagtgtt cagacttctc cacgaacaca
     1441 aaggtaaaaa agcacgctta gattggaata ctgatgctgc gtctttgatt ggagaagaac
     1501 ttcaagtaga tttcctggat catgttcccc tcacaacaca caactttgct cggaagacgt
     1561 tcctgaagct tgccttctgt gacatctgtc agaaattcct gctcaatgga tttcgatgtc
     1621 agacttgtgg ctacaaattt catgagcact gtagcaccaa agtacctact atgtgtgtgg
     1681 actggagtta atcaactttc ttgtacaaag tggtgatatc gctagctaga tagctagaag
     1741 cttgcggccg cacagctgta tacacgtgca agccagccag aactcgctcc tgaagaccca
     1801 gaggatctcg agcaccacca ccaccaccac taatgttaat taagttgggc gttcctaggc
     1861 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     1921 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     1981 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     2041 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     2101 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     2161 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     2221 ttgtttattt ttctaaatac attcaaatat gtatccgctg agcaataact agcataaccc
     2281 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2341 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2401 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2461 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2521 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2581 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2641 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2701 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2761 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga
     2821 tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2881 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2941 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3001 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3061 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3121 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3181 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3241 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3301 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3361 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3421 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3481 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3541 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3601 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3661 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3721 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3781 catactcttc ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg
     3841 gatacatatt tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa
     3901 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3961 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4021 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4081 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4141 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4201 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4261 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4321 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4381 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4441 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4501 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4561 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4621 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4681 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4741 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     4801 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4861 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4921 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4981 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5041 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5101 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5161 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5221 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5281 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5341 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5401 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5461 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5521 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5581 ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca
     5641 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5701 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5761 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5821 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5881 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5941 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6001 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6061 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6121 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6181 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6241 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6301 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6361 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6421 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6481 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6541 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6601 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6661 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6721 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6781 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6841 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6901 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6961 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     7021 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     7081 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     7141 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     7201 gcgaaa
//
