LOCUS       sequence-445628-        5292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of RalA (G23C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5292)
  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 5292)
  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 RalA(G23C) CL
FEATURES             Location/Qualifiers
     source          1..5292
                     /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(982..999)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1152..1238
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1330..1357
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1376..1467
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1468..1536,1537..2328)
                     /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(1686..1705)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2499..3087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2988..3007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3241..3258
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3335..3763)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3445..3466)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3657..3676
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3801..3820)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3993..4015
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4093..4170
                     /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             4171..5253
                     /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(4190..4209)
                     /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 atggcactag
      361 ctttacacaa agtaataatg gtgggctcgt gcggtgttgg caaatctgcg ttgaccctgc
      421 aatttatgta tgatgagttc gtggaggact acgagccgac gaaagctgac agctaccgca
      481 aaaaggtggt tctggatggt gaagaggtgc agattgatat cctcgacacc gcgggtcaag
      541 aagactacgc agcgattcgt gacaactatt tccgtagcgg tgaaggcttt ctgagcgttt
      601 tcagcattac cgaaatggaa tcctttgccg cgaccgcaga cttccgcgaa cagatcctga
      661 gagtcaaaga agacgagaac gttccgtttt tgctggttgg caacaaatct gatttggaag
      721 acaagcgtca ggttagcgtc gaggaggcaa agaaccgtgc ggaacaatgg aatgtaaatt
      781 atgttgagac ttccgctaag acccgtgcca atgtggataa ggtgttcttc gatctgatgc
      841 gtgagatccg cgcgcgttaa gaattcaaag gcctacgtcg acgagctcac tagtcgcggc
      901 cgctttcgaa tctagagcct gcagtctcga ggcatgcggt accaagcttg gctgttttgg
      961 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     1021 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     1081 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     1141 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1201 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     1261 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     1321 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt
     1381 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1441 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1501 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1561 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1621 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1681 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     1741 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1801 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1861 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1921 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1981 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     2041 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2101 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2161 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2221 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2281 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2341 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2401 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2461 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2521 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2581 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2641 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2701 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2761 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2821 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2881 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2941 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3001 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3061 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3121 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3181 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3241 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3301 ctgtgcggta tttcacaccg cataattttg ttaaaattcg cgttaaattt ttgttaaatc
     3361 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3421 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3481 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3541 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3601 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3661 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     3721 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     3781 ctgctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtaccag
     3841 tcacgtagcg atatcgctac gtgactggta cactccgcta tcgctacgtg actgggtcat
     3901 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3961 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4021 accgtcatca ccgaaacgcg cgaggcagca gatcaattcg cgcgcgaagg cgaagcggca
     4081 tgcatttacg ttgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc
     4141 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     4201 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     4261 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     4321 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     4381 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     4441 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     4501 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     4561 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     4621 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     4681 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     4741 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     4801 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     4861 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     4921 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     4981 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     5041 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     5101 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     5161 gcacccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5221 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagcg
     5281 cgaattgatc tg
//
