LOCUS       sequence-445633-        5346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of Rit1 (G47C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5346)
  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 5346)
  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 Rit1(G47C) CL
FEATURES             Location/Qualifiers
     source          1..5346
                     /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(1036..1053)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1206..1292
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1384..1411
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1430..1521
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1522..1590,1591..2382)
                     /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(1740..1759)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2553..3141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3042..3061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3295..3312
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3389..3817)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3499..3520)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3711..3730
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3855..3874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4047..4069
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4147..4224
                     /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             4225..5307
                     /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(4244..4263)
                     /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 tcaagggaat
      361 ataaactagt aatgttagga gcgtgcggtg tgggcaagtc cgccatgacc atgcaattta
      421 tcagccatcg ctttccggaa gatcatgatc cgacgatcga agatgcatat aaaatccgta
      481 ttcgaatcga tgacgaaccg gcaaatctgg acatcttaga caccgccggt caagcagaat
      541 ttacggcgat gcgtgaccag tacatgagag ctggtgaggg ttttattatt tgctactcga
      601 tcaccgaccg tcgtagcttc cacgaagtcc gcgaattcaa gcagctgatt taccgcgtgc
      661 gccgtaccga cgacaccccg gttgttttgg ttggcaacaa aagtgatctg aaacaactcc
      721 gccaggtcac caaagaggaa ggcctggcgc tggcgcgtga gttcagctgt ccgttcttcg
      781 agacttctgc ggcttaccgt tattatatcg acgatgtttt tcacgctctg gtgcgtgaga
      841 ttcgtcgtaa agagaaggag gcggtgttgg cgatggagaa aaagtccaag ccaaaaaaca
      901 gcgtgtggaa gtaagaattc aaaggcctac gtcgacgagc tcactagtcg cggccgcttt
      961 cgaatctaga gcctgcagtc tcgaggcatg cggtaccaag cttggctgtt ttggcggatg
     1021 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
     1081 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     1141 gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
     1201 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     1261 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     1321 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     1381 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt
     1441 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1501 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1561 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1621 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1681 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1741 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     1801 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1861 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     1921 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1981 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2041 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     2101 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2161 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2221 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2281 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2341 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2401 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2461 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2521 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2581 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2641 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2701 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2761 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2821 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2881 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2941 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3001 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3061 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3121 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3181 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3241 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3301 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3361 ggtatttcac accgcataat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3421 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3481 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3541 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3601 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3661 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3721 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3781 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcta
     3841 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta ccagtcacgt
     3901 agcgatatcg ctacgtgact ggtacactcc gctatcgcta cgtgactggg tcatggctgc
     3961 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4021 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4081 atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc ggcatgcatt
     4141 tacgttgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     4201 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     4261 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     4321 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     4381 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     4441 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     4501 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     4561 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     4621 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     4681 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     4741 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     4801 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     4861 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     4921 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     4981 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     5041 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     5101 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     5161 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcaccc
     5221 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5281 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt
     5341 gatctg
//
