LOCUS       sequence-170056-        6767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the RAS-binding domain of RAF1 for in vitro analysis of 
            RAS activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6767)
  TITLE     FNLCR RAS Initiative
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6767)
  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: 
            "Unpublished"
COMMENT     Sequence Label: GST-thr-RAF1-RBD
FEATURES             Location/Qualifiers
     source          1..6767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      16..604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     505..524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     758..775
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    790..929
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(855..874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1015..1037
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1031..1222)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1794..2876)
                     /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     2838..2857
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2877..2954)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3160..3179
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3267..3286
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3267..3285
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3286..3310
                     /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    3361..3385
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3411..4064
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             4071..4088
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    complement(4564..4588)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4600..4632
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             4639..4656
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4822..4908
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5000..5027
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5081..5099)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5095..5142
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5179..5634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5266..5285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5476..5497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5749..6540,6541..6609))
                     /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     6372..6391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6610..6702)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
       61 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      121 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
      181 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      241 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      301 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      361 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
      421 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
      481 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
      541 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
      601 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
      661 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
      721 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      781 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
      841 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
      901 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
      961 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     1021 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     1081 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     1141 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     1201 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     1261 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     1321 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     1381 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     1441 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     1501 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     1561 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     1621 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     1681 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1741 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1801 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1861 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     1921 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     1981 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     2041 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     2101 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     2161 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     2221 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     2281 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     2341 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     2401 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     2461 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     2521 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     2581 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     2641 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     2701 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     2761 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     2821 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     2881 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     2941 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     3001 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     3061 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     3121 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     3181 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     3241 cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     3301 taacaattcc cctctagcta gatagctaga agcttgcggc cgcacagctg tatacacatc
     3361 acaagtttgt acaaaaaagt tggctttaac tttaagaagg agatatacat atgagtccta
     3421 tcttgggtta ctggaagatt aaaggtttgg tccaaccaac gcgtctgttg ctggagtacc
     3481 tggaagaaaa gtatgaagaa cacctgtacg agcgtgacga aggcgacaaa tggcgcaata
     3541 aaaagttcga gctgggcctg gagttcccga acctcccgta ttacatcgac ggtgatgtta
     3601 aactgacgca gtctatggct atcattcgtt acattgcgga caagcataac atgctgggtg
     3661 gctgcccaaa agagcgcgct gagatttcca tgttggaagg tgcggtgctg gatattcgtt
     3721 atggtgttag ccgtattgcg tatagcaaag attttgaaac gctgaaggtt gactttctga
     3781 gcaagctgcc tgagatgctg aagatgttcg aggatcgcct gtgccacaag acttacctga
     3841 atggcgacca cgtgacccac ccggacttca tgctttatga cgcactggat gtcgtgctgt
     3901 acatggaccc gatgtgtctg gacgcctttc cgaaactggt ctgcttcaag aagcgcatcg
     3961 aagcgatccc gcagatcgat aagtacctga aaagctctaa gtatatcgca tggccgctgc
     4021 agggctggca agccaccttc ggtggtggtg accatccgcc gaaaagcgac ttggttccgc
     4081 gtggttcgcc gattcacatt atggaacata tccagggcgc atggaaaact atcagcaatg
     4141 gcttcggttt caaagatgcg gtgtttgatg gctccagctg cattagcccg accatcgtac
     4201 agcagttcgg ttaccagcgt cgtgcgagcg atgatggcaa actgaccgat ccgagcaaga
     4261 ccagcaacac gattcgcgtt tttctgccga ataaacaacg caccgtcgtc aacgttcgta
     4321 acggtatgag cctgcacgat tgtctgatga aggctctgaa agtgcgcggt ctgcaaccgg
     4381 agtgttgcgc agtttttcgt ctgttacacg agcacaaggg taaaaaggcc cgtctggact
     4441 ggaataccga cgcggccagc ctgattggcg aggaactgca agtcgatttt ctggatcatg
     4501 tgccgctgac gacccacaac ttcgcgcgta aaaccttttt gaagctgggt atccatcgtg
     4561 actaatcaac tttcttgtac aaagtggtga tgtgcaagcc agccagaact cgctcctgaa
     4621 gacccagagg atctcgagca ccaccaccac caccactaat gttaattaag ttgggcgttc
     4681 ctaggctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca
     4741 tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga
     4801 gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     4861 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     4921 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     4981 gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa
     5041 actcttttgt ttatttttct aaatacattc aaatatgtat ccgctgagca ataactagca
     5101 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     5161 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5221 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5281 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5341 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5401 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5461 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5521 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5581 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttctggcg
     5641 gcacgatggc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5701 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5761 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5821 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5881 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5941 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6001 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6061 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6121 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6181 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6241 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6301 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6361 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6421 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6481 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6541 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6601 aatactcata ctcttccttt ttcaatcatg attgaagcat ttatcagggt tattgtctca
     6661 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggtcat gaccaaaatc
     6721 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaa
//
