LOCUS       sequence-313755-        6814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli protein expression of His6-Halotag-tev-Hs.RAF1(52-131).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6814)
  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 6814)
  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: R726-M01-892: His6-Halotag-tev-Hs.RAF1(52-131)
FEATURES             Location/Qualifiers
     source          1..6814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             57..89
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             96..113
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      279..365
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      457..484
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(538..556)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      552..599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      636..1091
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(723..742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     933..954
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1206..1997,1998..2066))
                     /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     1829..1848
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2067..2159)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2240..2828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2729..2748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2982..2999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3014..3153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3079..3098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3239..3261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3255..3446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4018..5100)
                     /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     5062..5081
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5101..5178)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    5279..5299
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     5325..5344
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5432..5451
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5432..5450
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5451..5475
                     /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)."
     protein_bind    complement(5491..5511)
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             5547..5564
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5565..6455
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             6468..6488
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    6504..6528
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6528..6548
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     protein_bind    complement(6787..6811)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 atcgctagct agatagctag aagcttgcgg ccgcacagct gtatacacgt gcaagccagc
       61 cagaactcgc tcctgaagac ccagaggatc tcgagcacca ccaccaccac cactaatgtt
      121 aattaagttg ggcgttccta ggctgataaa acagaatttg cctggcggca gtagcgcggt
      181 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
      241 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
      301 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
      361 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
      421 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
      481 tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg
      541 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      601 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
      661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      841 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      901 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      961 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1021 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1081 gtttacaatt tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc
     1141 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     1201 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     1261 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     1321 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     1381 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1441 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1501 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1561 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     1621 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     1681 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     1741 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     1801 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     1861 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     1921 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     1981 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2041 gcgacacgga aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta
     2101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2161 aggtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2221 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2281 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2341 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2401 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2461 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2521 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2581 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2641 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2701 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2761 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2821 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2881 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2941 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3001 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3061 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     3121 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3181 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3241 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3301 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3361 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3421 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3481 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3541 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     3601 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3661 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3721 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3781 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3841 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3901 atgctagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     3961 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4021 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4081 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4141 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4201 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4261 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     4321 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4381 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4441 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4501 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4561 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4621 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4681 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4741 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4801 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4861 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4921 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4981 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5041 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5101 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5161 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5221 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgatcaca
     5281 actttgtata gaaaagttgg ctggcgagcc cgatcttccc catcggtgat gtcggcgata
     5341 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5401 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     5461 gcggataaca attcccctct agaaataagc caacttttgt atacaaagtg gtcatttaac
     5521 tttaagaagg agatatacat atgggccacc atcaccatca ccatatggca gaaatcggta
     5581 ctggctttcc attcgacccc cattatgtgg aagtcctggg cgagcgcatg cactacgtcg
     5641 atgttggtcc gcgcgatggc acccctgtgc tgttcctgca cggtaacccg acctcctcct
     5701 acgtgtggcg caacatcatc ccgcatgttg caccgaccca tcgctgcatt gctccagacc
     5761 tgatcggtat gggcaaatcc gacaaaccag acctgggtta tttcttcgac gaccacgtcc
     5821 gcttcatgga tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg
     5881 actggggctc cgctctgggt ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta
     5941 ttgcatttat ggagttcatc cgccctatcc cgacctggga cgaatggcca gaatttgccc
     6001 gcgagacctt ccaggccttc cgcaccaccg acgtcggccg caagctgatc atcgatcaga
     6061 acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga
     6121 tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc
     6181 caaacgagct gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca
     6241 tggactggct gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc
     6301 tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg
     6361 acatcggccc gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga
     6421 tcgcgcgctg gctgtcgacg ctcgagattt ccggcgagcc aaccactgag gatctgtact
     6481 ttcagagcga taacgcgatc gccacaactt tgtacaaaaa agttggcgag aacctgtact
     6541 ttcagtccaa gaccagcaac acgatccgcg tgttcctgcc gaataagcaa cgtaccgtcg
     6601 ttaatgtgcg caacggtatg agcctgcatg actgcctgat gaaagcactg aaagtgcgtg
     6661 gtctgcagcc ggagtgttgt gcggtctttc gtctgctgca cgagcataag ggcaaaaaag
     6721 cccgtctgga ctggaatacc gatgcagcga gcctgattgg tgaagaactg caagtcgact
     6781 tcttgtaatc aactttcttg tacaaagttg tgat
//
