LOCUS       sequence-445635-        5367 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Protein purification of M-Ras (G22C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5367)
  AUTHORS   Morstein J, Bowcut V, Fernando M, Yang Y, Zhu L, Jenkins ML, Evans 
            JT, Guiley KZ, Peacock DM, Krahnke S, Lin Z, Taran KA, Huang BJ, 
            Stephen AG, Burke JE, Lightstone FC, Shokat KM
  TITLE     Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic
            pocket.
  JOURNAL   Cell. 2024 Sep 6:S0092-8674(24)00908-5. doi: 
            10.1016/j.cell.2024.08.017.
  PUBMED    39255801
REFERENCE   2  (bases 1 to 5367)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2024 
            Sep 6:S0092-8674(24)00908-5. doi: 10.1016/j.cell.2024.08.017."
COMMENT     Sequence Label: pProEx M-Ras(G22C) CL
FEATURES             Location/Qualifiers
     source          1..5367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    230..246
                     /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)."
     primer_bind     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             279..296
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..338
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1057..1074)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1227..1313
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1405..1432
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1451..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3410..3838)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3541)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3732..3751
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3876..3895)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4068..4090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4168..4245
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4246..5328
                     /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     complement(4265..4284)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gtccgtataa tctgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagaccatgt cgtactacca tcaccatcac catcacgatt
      301 acgatatccc aacgaccgaa aacctgtatt ttcagggcgc catgggatcc atggcaacaa
      361 gtgctgtacc ctcagataat cttccgacct ataaactcgt cgtggtgggc gattgcggcg
      421 tcggcaagtc cgcgctgact atccagtttt tccaaaagat cttcgtgccg gattacgacc
      481 cgacgatcga ggatagctac ctgaaacaca ccgaaatcga caaccagtgg gcaattttgg
      541 acgttttgga taccgcaggt caggaggagt tctctgctat gcgtgaacaa tatatgcgca
      601 ccggtgacgg ctttctgatc gtgtacagcg ttaccgataa ggccagcttt gaacatgttg
      661 acagattcca ccagctgatt ctgcgcgtga aagaccgcga gagcttcccg atgattttgg
      721 tggctaacaa agtagacctg atgcatctgc gtaaaatcac ccgtgagcag ggtaaagaaa
      781 tggcgacgaa gcacaacatt ccgtatatcg agacgtccgc gaaagatccg ccactgaatg
      841 ttgacaaagc gtttcacgat ttagttcgtg ttattcgtca gcaaattccg gaaaaatcgc
      901 aaaaaaagaa gaagaagacc aagtggcgtg gttaagaatt caaaggccta cgtcgacgag
      961 ctcactagtc gcggccgctt tcgaatctag agcctgcagt ctcgaggcat gcggtaccaa
     1021 gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg
     1081 cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga
     1141 ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca
     1201 tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     1261 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     1321 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     1381 aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc
     1441 tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3361 tttctcctta cgcatctgtg cggtatttca caccgcataa ttttgttaaa attcgcgtta
     3421 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3481 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3541 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3601 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3661 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3721 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3781 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     3841 cattcgccat tcaggctgct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3901 ttaagccagt accagtcacg tagcgatatc gctacgtgac tggtacactc cgctatcgct
     3961 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4021 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4081 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagcagatca attcgcgcgc
     4141 gaaggcgaag cggcatgcat ttacgttgac accatcgaat ggtgcaaaac ctttcgcggt
     4201 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     4261 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     4321 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     4381 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     4441 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     4501 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     4561 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     4621 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     4681 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     4741 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     4801 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     4861 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     4921 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     4981 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     5041 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     5101 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     5161 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     5221 agaaaaacca ccctggcacc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5281 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5341 taatgtaagt tagcgcgaat tgatctg
//
