LOCUS       sequence-455362-        5316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of Rac1 (G12C, K96H).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5316)
  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 5316)
  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: "Cell. 2024 
            Sep 6:S0092-8674(24)00908-5. doi: 10.1016/j.cell.2024.08.017."
COMMENT     Sequence Label: pProEx Rac1(G12C, K96H) CL
FEATURES             Location/Qualifiers
     source          1..5316
                     /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(1006..1023)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1176..1262
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1354..1381
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1400..1491
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1492..1560,1561..2352)
                     /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(1710..1729)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2523..3111
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3012..3031
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3265..3282
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3359..3787)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3469..3490)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3681..3700
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3825..3844)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4017..4039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4117..4194
                     /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             4195..5277
                     /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(4214..4233)
                     /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 atgcaggcca
      361 tcaagagtgt ggtggtggga gactgtgctg taggtaaaac tagcctactg atcagttaca
      421 caaccaatgc atttcctgga gaatatatcc ctactgtctt tgacaattat tctgccaatg
      481 ttatggtaga tggaaaaccg gtgaatctgg gcttatggga tacagctgga caagaagatt
      541 atgacagatt acgcccccta tcctatccgc aaacagatgt gttcttaatt agcttttccc
      601 ttgtgagtcc tgcatcattt gaaaatgtcc gtgcacattg gtatcctgag gtgcggcacc
      661 acagtcccaa cactcccatc atcctagtgg gaactaaact tgatcttagg gatgataaag
      721 acacgatcga gaaactgaag gagaagaagc tgactcccat cacctatccg cagggtctag
      781 ccatggctaa ggagattggt gctgtaaaat acctggagag ctcggcgctc acacagcgag
      841 gcctcaagac agtgtttgac gaagcgatcc gagcagtcct ctgagaattc aaaggcctac
      901 gtcgacgagc tcactagtcg cggccgcttt cgaatctaga gcctgcagtc tcgaggcatg
      961 cggtaccaag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     1021 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     1081 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
     1141 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
     1201 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     1261 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     1321 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt
     1381 ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1441 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1501 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1561 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1621 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1681 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     1741 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     1801 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     1861 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     1921 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     1981 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2041 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2101 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2161 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2221 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2281 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2341 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2401 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2461 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2521 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2581 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2641 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2701 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2761 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2821 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2881 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2941 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3001 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3061 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3121 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3181 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3241 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3301 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataat tttgttaaaa
     3361 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3421 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3481 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3541 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3601 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3661 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3721 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3781 ggcgcgtccc attcgccatt caggctgcta tggtgcactc tcagtacaat ctgctctgat
     3841 gccgcatagt taagccagta ccagtcacgt agcgatatcg ctacgtgact ggtacactcc
     3901 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3961 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4021 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa
     4081 ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc
     4141 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     4201 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     4261 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     4321 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     4381 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     4441 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     4501 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     4561 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     4621 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     4681 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     4741 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     4801 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     4861 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     4921 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     4981 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     5041 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     5101 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     5161 ctggtgaaaa gaaaaaccac cctggcaccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5221 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     5281 caacgcaatt aatgtaagtt agcgcgaatt gatctg
//
