LOCUS       sequence-445631-        5331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of Ypt1 (S17C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5331)
  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 5331)
  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 Ypt1(S17C) CL
FEATURES             Location/Qualifiers
     source          1..5331
                     /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(1021..1038)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1191..1277
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1369..1396
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1415..1506
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1507..1575,1576..2367)
                     /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(1725..1744)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2538..3126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3027..3046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3280..3297
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3374..3802)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3484..3505)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3696..3715
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3840..3859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4032..4054
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4132..4209
                     /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             4210..5292
                     /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(4229..4248)
                     /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 atgaattcag
      361 aatatgatta cctatttaaa ttgctcctta ttggcaactg cggtgtcggt aaaagctgtc
      421 tgctgttacg tttctccgat gacacctata ccaacgatta catcagcacc atcggtgtcg
      481 atttcaaaat caagaccgtg gagctggacg gcaagacggt taaactgcaa atctgggata
      541 ctgcgggtca agagcgcttt cgtaccatta cctctagcta ttaccgcggc agtcacggga
      601 ttatcattgt ttacgacgtg accgatcagg agtcgttcaa cggtgtgaag atgtggctgc
      661 aagaaattga tcgttatgct acctctacgg ttctgaagtt gttggttggc aacaagtgcg
      721 atctgaagga caagcgtgtt gttgagtacg acgtggccaa agaatttgcg gatgcaaata
      781 agatgccgtt tctggaaacc tccgcgctgg acagcaccaa cgtagaagac gctttcctga
      841 cgatggcgag acagatcaaa gagtccatga gccagcagaa tttgaatgaa acttaatgag
      901 aattcaaagg cctacgtcga cgagctcact agtcgcggcc gctttcgaat ctagagcctg
      961 cagtctcgag gcatgcggta ccaagcttgg ctgttttggc ggatgagaga agattttcag
     1021 cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg
     1081 cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc
     1141 cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac
     1201 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1261 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     1321 ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc
     1381 tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca
     1441 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1501 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1561 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     1621 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     1681 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     1741 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     1801 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     1861 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     1921 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     1981 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2041 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2101 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2161 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2221 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2281 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2341 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2401 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2461 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2521 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2581 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2641 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2701 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2761 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2821 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2881 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2941 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3001 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3061 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3121 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3181 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3241 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3301 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3361 ataattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3421 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3481 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3541 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3601 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3661 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3721 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3781 aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgctatggtg cactctcagt
     3841 acaatctgct ctgatgccgc atagttaagc cagtaccagt cacgtagcga tatcgctacg
     3901 tgactggtac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3961 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4021 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4081 gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc
     4141 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     4201 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     4261 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     4321 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     4381 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     4441 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     4501 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     4561 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     4621 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     4681 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     4741 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     4801 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     4861 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     4921 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     4981 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     5041 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     5101 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     5161 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cacccaatac gcaaaccgcc
     5221 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     5281 agcgggcagt gagcgcaacg caattaatgt aagttagcgc gaattgatct g
//
