LOCUS       sequence-445637-        5325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein purification of RhoA (G14C).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5325)
  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 5325)
  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 RhoA(G14C) CL
FEATURES             Location/Qualifiers
     source          1..5325
                     /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(1015..1032)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1185..1271
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1363..1390
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1409..1500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1501..1569,1570..2361)
                     /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(1719..1738)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2532..3120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3021..3040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3274..3291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3368..3796)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3478..3499)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3690..3709
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3834..3853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4026..4048
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4126..4203
                     /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             4204..5286
                     /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(4223..4242)
                     /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 atggctgcaa
      361 ttaggaagaa actagtaata gtgggcgatt gcgcgagcgg taagacgagc ttgctgattg
      421 ttttctccaa agaccagttc ccggaggtgt acgtgccaac ggtcttcgag aactatgtgg
      481 ccgacatcga agtggatggc aagcaagttg aattggctct gtgggatact gcgggtcagg
      541 aggactatga tcgtctgcgc cctctgtcct acccggacac cgatgttatt ctgatgagct
      601 tttctattga ctcgccggac agcctggaaa acatcccgga gaagtggacc ccggaggtca
      661 aacatttttc tccgaatgtt ccgattatct tggttggtaa caaaaaggac ctgcgtaatg
      721 atgaacacac ccgtcgtgag cttgccaaga tgaaacaaga gccggttaag ccggaggaag
      781 gtcgtgatat ggcgaaccgc atcggcgcat ttggttacat ggaaagcagc gcaaaaacca
      841 aagacggcgt tcgtgaagtg ttcgaaatgg cgacccgcgc tgcgctgcag taagaattca
      901 aaggcctacg tcgacgagct cactagtcgc ggccgctttc gaatctagag cctgcagtct
      961 cgaggcatgc ggtaccaagc ttggctgttt tggcggatga gagaagattt tcagcctgat
     1021 acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag
     1081 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     1141 tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg
     1201 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     1261 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     1321 gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg
     1381 atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg
     1441 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1501 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1561 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1621 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1681 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1741 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1801 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1861 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1921 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1981 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2041 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2101 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2161 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2221 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2281 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2341 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2401 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2461 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2521 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2581 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2641 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     2701 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2761 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2821 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2881 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2941 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3001 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3061 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3121 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3181 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3241 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3301 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataatt
     3361 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3421 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3481 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     3541 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     3601 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3661 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3721 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3781 ccgctacagg gcgcgtccca ttcgccattc aggctgctat ggtgcactct cagtacaatc
     3841 tgctctgatg ccgcatagtt aagccagtac cagtcacgta gcgatatcgc tacgtgactg
     3901 gtacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3961 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4021 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4081 gcagatcaat tcgcgcgcga aggcgaagcg gcatgcattt acgttgacac catcgaatgg
     4141 tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     4201 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     4261 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     4321 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     4381 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     4441 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     4501 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     4561 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     4621 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     4681 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     4741 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     4801 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     4861 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     4921 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     4981 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     5041 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     5101 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     5161 cccgtctcac tggtgaaaag aaaaaccacc ctggcaccca atacgcaaac cgcctctccc
     5221 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5281 cagtgagcgc aacgcaatta atgtaagtta gcgcgaattg atctg
//
