LOCUS       sequence-313753-        6957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Hs.RAF1(136-188).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6957)
  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 6957)
  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-X45-566: His6-MBP-tev-Hs.RAF1(136-188)
FEATURES             Location/Qualifiers
     source          1..6957
                     /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"
     protein_bind    complement(1441..1465)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1525..1557
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1564..1581
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1747..1833
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1925..1952
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2006..2024)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2020..2067
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2104..2559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2191..2210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2401..2422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2674..3465,3466..3534))
                     /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     3297..3316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3535..3627)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3708..4296
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4197..4216
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4450..4467
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4482..4621
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4547..4566)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4707..4729
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4723..4914)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5486..6568)
                     /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     6530..6549
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6569..6646)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6852..6871
                     /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 aaaatctgta ttttcaaggc ttgaccaccc ataacttcgc gcgtaagacc ttcctgaaac
     1321 tggcattttg cgacatctgt cagaaattcc tgctgaacgg tttccgctgc cagacgtgtg
     1381 gttacaagtt tcacgagcac tgcagcacga aagttccgac tatgtgtgtg gattggtcct
     1441 aatcaacttt cttgtacaaa gtggtgatat cgctagctag atagctagaa gcttgcggcc
     1501 gcacagctgt atacacgtgc aagccagcca gaactcgctc ctgaagaccc agaggatctc
     1561 gagcaccacc accaccacca ctaatgttaa ttaagttggg cgttcctagg ctgataaaac
     1621 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     1681 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     1741 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     1801 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     1861 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     1921 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     1981 tttctaaata cattcaaata tgtatccgct gagcaataac tagcataacc ccttggggcc
     2041 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2101 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2161 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2221 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2281 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2341 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2401 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2461 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2521 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag
     2581 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2641 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2701 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2761 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2821 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2881 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2941 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3001 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3061 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3121 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3181 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3241 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3301 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3361 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3421 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3481 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3541 cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat
     3601 ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt
     3661 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3721 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3781 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3841 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3901 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3961 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4021 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4081 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4141 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4201 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4261 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4321 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4381 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4441 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4501 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4561 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4621 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4681 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4741 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4801 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4861 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4921 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4981 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5041 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5101 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5161 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5221 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5281 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5341 ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga
     5401 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5461 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5521 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5581 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5641 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5701 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5761 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5821 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5881 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5941 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     6001 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     6061 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     6121 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     6181 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     6241 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     6301 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     6361 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6421 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6481 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6541 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6601 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6661 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6721 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6781 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6841 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6901 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaa
//
