LOCUS       sequence-313416-        7218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-MBP-tev-Hs.RAF1(52-192).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  AUTHORS   Tran TH, Chan AH, Young LC, Bindu L, Neale C, Messing S, Dharmaiah 
            S, Taylor T, Denson JP, Esposito D, Nissley DV, Stephen AG, 
            McCormick F, Simanshu DK
  TITLE     KRAS interaction with RAF1 RAS-binding domain and cysteine-rich 
            domain provides insights into RAS-mediated RAF activation.
  JOURNAL   Nat Commun. 2021 Feb 19;12(1):1176. doi: 10.1038/s41467-021-21422-x.
  PUBMED    33608534
REFERENCE   2  (bases 1 to 7218)
  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: "Nat 
            Commun."; date: "2021-02-19"; volume: "12(1):1176. doi"; pages: " 
            10.1038/s41467-021-21422-x"
COMMENT     Sequence Label: R717-X61-566: His6-MBP-tev-Hs.RAF1(52-192)
FEATURES             Location/Qualifiers
     source          1..7218
                     /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(1702..1726)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1786..1818
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1825..1842
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2008..2094
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2186..2213
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2267..2285)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2281..2328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2365..2820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2452..2471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2662..2683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2935..3726,3727..3795))
                     /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     3558..3577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3796..3888)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3969..4557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4458..4477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4711..4728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4743..4882
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4808..4827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4968..4990
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4984..5175)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5747..6829)
                     /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     6791..6810
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6830..6907)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7113..7132
                     /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 actggagtaa catcagacaa taatcaactt tcttgtacaa agtggtgata tcgctagcta
     1741 gatagctaga agcttgcggc cgcacagctg tatacacgtg caagccagcc agaactcgct
     1801 cctgaagacc cagaggatct cgagcaccac caccaccacc actaatgtta attaagttgg
     1861 gcgttcctag gctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
     1921 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc
     1981 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     2041 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     2101 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     2161 taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt
     2221 ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc tgagcaataa
     2281 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2341 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2401 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2461 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2521 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2581 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2641 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2701 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2761 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     2821 ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2881 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2941 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3001 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3061 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3121 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3181 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3241 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3301 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3361 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3421 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3481 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     3541 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     3601 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     3661 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3721 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3781 atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat cagggttatt
     3841 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggtcatgacc
     3901 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3961 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4021 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4081 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4141 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4201 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4261 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4321 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4381 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4441 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4501 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4561 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4621 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4681 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4741 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4801 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4861 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4921 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4981 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5041 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5101 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5161 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5221 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5281 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5341 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5401 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5461 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5521 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5581 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat
     5641 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5701 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5761 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5821 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5881 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5941 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     6001 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     6061 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     6121 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     6181 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6241 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     6301 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     6361 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     6421 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     6481 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     6541 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6601 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6661 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6721 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6781 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6841 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     6901 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6961 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     7021 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     7081 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     7141 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     7201 gatctcgatc ccgcgaaa
//
