LOCUS       sequence-215108-        7245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of RALA_Ras.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7245)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7245)
  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: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_RALA_Ras
FEATURES             Location/Qualifiers
     source          1..7245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1440..1487
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3080..3535)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3217..3238)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3429..3448
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3577..3599)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3685..3825
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3740..3759
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3840..3857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4011..4598)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4769..5560,5561..5629))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5392..5411
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5630..5734)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5802..5820
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5843..5920
                     /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             5921..7003
                     /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(5940..5959)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccagcct ggcactgcat aaagttatta tggttggtag cggtggtgtt ggtaaaagcg
      961 cactgaccct gcagtttatg tatgatgaat tcgtggaaga ttacgaaccg accaaagcag
     1021 atagttatcg taaaaaagtt gtgctggatg gtgaagaagt gcagattgat attctggata
     1081 ccgcaggtca agaggattat gcagcaattc gtgataatta ctttcgcagc ggtgaaggtt
     1141 ttctgtgcgt ttttagcatt accgaaatgg aaagctttgc agcaaccgca gattttcgtg
     1201 agcagattct gcgtgttaaa gaagatgaaa acgttccgtt tctgctggtt ggcaataaaa
     1261 gcgatctgga agataaacgt caggtgagcg ttgaagaagc aaaaaatcgc gcagaacagt
     1321 ggaatgtgaa ttatgttgaa accagcgcaa aaacccgtgc caatgttgat aaagtgtttt
     1381 ttgatctgat gcgcgaaatt cgtgcacgca aaatggaaga tagtgcggcc gcataataaa
     1441 ccgatacaat taaaggctcc ttttggagcc ttttttttgg agattttcaa cgtgaaaaaa
     1501 ttattattcg caattccttt agttgttcct ttctattctc actccgctga aactgttgaa
     1561 agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa
     1621 actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt
     1681 tgtactggtg acgaaactca gtgtctagct agagtggcgg tggctctggt tccggtgatt
     1741 ttgattatga aaagatggca aacgctaata agggggctat gaccgaaaat gccgatgaaa
     1801 acgcgctaca gtctgacgct aaaggcaaac ttgattctgt cgctactgat tacggtgctg
     1861 ctatcgatgg tttcattggt gacgtttccg gccttgctaa tggtaatggt gctactggtg
     1921 attttgctgg ctctaattcc caaatggctc aagtcggtga cggtgataat tcacctttaa
     1981 tgaataattt ccgtcaatat ttaccttccc tccctcaatc ggttgaatgt cgcccttttg
     2041 tctttggcgc tggtaaacca tatgaatttt ctattgattg tgacaaaata aacttattcc
     2101 gtggtgtctt tgcgtttctt ttatatgttg ccacctttat gtatgtattt tctacgtttg
     2161 ctaacatact gcgtaataag gagtcttaat catgccagtt cttttggcta gcgccgccct
     2221 ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg
     2281 aatggaagcc ggcggcacct cgctaacgga ttcaccactc caagaattgg agccaatcaa
     2341 ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc agaacatatc catcgcgtcc
     2401 gccatctcca gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg
     2461 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     2521 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     2581 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     2641 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     2701 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     2761 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     2821 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     2881 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     2941 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     3001 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     3061 ctttaccgca ggatccgcga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3121 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3181 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3241 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3301 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3361 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3421 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3481 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgg
     3541 atcctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3601 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3661 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3721 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     3781 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     3841 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3901 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3961 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4021 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4081 acaggactat aaagatacca gggtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4141 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4201 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4261 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4321 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4381 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4441 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4501 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4561 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4621 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4681 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4741 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4801 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4861 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4921 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4981 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5041 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
     5101 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5161 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5221 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5281 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5341 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
     5401 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5461 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5521 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5581 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5641 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5701 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5761 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5821 cctttcgtct tcaagaattc ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     5881 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     5941 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     6001 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     6061 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     6121 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     6181 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     6241 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     6301 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     6361 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     6421 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     6481 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     6541 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     6601 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     6661 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     6721 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     6781 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     6841 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     6901 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     6961 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7021 taagttagct cactcattag gcacaattct catgtttgac agcttatcat cgactgcacg
     7081 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     7141 gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt
     7201 gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctg
//
