LOCUS       sequence-445630-        5289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of Rap1A (G12C, L96F).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5289)
  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 5289)
  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 Rap1A(G12C, L96F) CL
FEATURES             Location/Qualifiers
     source          1..5289
                     /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(979..996)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1149..1235
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1327..1354
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1373..1464
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1465..1533,1534..2325)
                     /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(1683..1702)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2496..3084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2985..3004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3238..3255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3332..3760)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3442..3463)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3654..3673
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3798..3817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3990..4012
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4090..4167
                     /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             4168..5250
                     /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(4187..4206)
                     /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 atgagggaat
      361 ataaattagt tgtactagga tcctgcggtg taggcaagtc cgcgttgacc gttcagttcg
      421 tgcagggcat tttcgtggag aaatacgacc cgaccattga agacagctat cgtaagcagg
      481 ttgaggtgga ttctcagcag agcatgctcg agatcctgga tacggcaggc accgagcaat
      541 ttacggctat gcgtgattta tatatgaaaa acggtcaagg ctttgccctg gtctacagca
      601 ttaccgcgca gtcgaccttc aacgatctgc aggatttcag agaacagatc ctgcgcgtga
      661 aggacactga ggacgttccg atgattctgg tcggtaataa gagtgatttg gaggacgaac
      721 gtgttgttgg taaagaacaa ggtcaaaacc tggcgcgtca atggagcaat agcgcgtttc
      781 tggaatctag cgctaaaagc aaaatcaacg tgaatgaaat cttctacgac ctggtgcgtc
      841 aaatcaaccg caagtaagaa ttcaaaggcc tacgtcgacg agctcactag tcgcggccgc
      901 tttcgaatct agagcctgca gtctcgaggc atgcggtacc aagcttggct gttttggcgg
      961 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     1021 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     1081 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     1141 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1201 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     1261 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     1321 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     1381 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1441 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     1501 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     1561 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     1621 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1681 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     1741 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1801 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1861 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     1921 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     1981 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     2041 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2101 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2161 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2221 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2281 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2341 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     2401 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     2461 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2521 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2581 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2641 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2701 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2761 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2821 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2881 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2941 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3001 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3061 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3121 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3181 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3241 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3301 tgcggtattt cacaccgcat aattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3361 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3421 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3481 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3541 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3601 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3661 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3721 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     3781 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtaccagtca
     3841 cgtagcgata tcgctacgtg actggtacac tccgctatcg ctacgtgact gggtcatggc
     3901 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3961 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4021 gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc gcgaaggcga agcggcatgc
     4081 atttacgttg acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg
     4141 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     4201 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4261 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4321 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4381 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4441 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4501 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4561 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4621 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4681 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4741 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4801 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     4861 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     4921 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4981 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     5041 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     5101 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggca
     5161 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5221 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagcgcga
     5281 attgatctg
//
