LOCUS       sequence-231647-        5212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  purification of GST-RAF1 RBD from E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5212)
  AUTHORS   Riecken LB, Tawamie H, Dornblut C, Buchert R, Ismayel A, Schulz A, 
            Schumacher J, Sticht H, Pohl KJ, Cui Y, Reis A, Morrison H, Abou 
            Jamra R
  TITLE     Inhibition of RAS activation due to a homozygous ezrin variant in 
            patients with profound intellectual disability.
  JOURNAL   Hum Mutat. 2015 Feb;36(2):270-8. doi: 10.1002/humu.22737.
  PUBMED    25504542
REFERENCE   2  (bases 1 to 5212)
  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"; doi: "10.1002/humu"; 
            journalName: "Hum Mutat"; date: "2015-02"; volume: "36"; issue: "2";
            pages: "270-8"
COMMENT     Sequence Label: pGEX-4T-1_RAF1 (51-131)
FEATURES             Location/Qualifiers
     source          1..5212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(34..39,40..55,56..62)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    70..86
                     /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)."
     CDS             109..762
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     720..742
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             769..786
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1113..1135)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1280..1298)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1366..1470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1471..1539,1540..2331)
                     /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     complement(1689..1708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2502..3090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2991..3010
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3244..3261
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3334..3411
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3412..4494
                     /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(3431..3450)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4543..4548,4549..4566,4567..4573)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4581..4597
                     /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     4605..4621
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4605..4621
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4617..5144
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4637..4654)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4637..4653)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4646..4668)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa
       61 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat gtcccctata
      121 ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt
      181 gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa
      241 aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa
      301 ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt
      361 tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac
      421 ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc
      481 aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat
      541 ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac
      601 atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa
      661 gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag
      721 ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct ggttccgcgt
      781 ggatcccctt ctaagacaag caacactatc cgtgttttct tgccgaacaa gcaaagaaca
      841 gtggtcaatg tgcgaaatgg aatgagcttg catgactgcc ttatgaaagc actcaaggtg
      901 aggggcctgc aaccagagtg ctgtgcagtg ttcagacttc tccacgaaca caaaggtaaa
      961 aaagcacgct tagattggaa tactgatgct gcgtctttga ttggagaaga acttcaagta
     1021 gatttcctgt aggaattccc gggtcgactc gagcggccgc atcgtgactg actgacgatc
     1081 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     1141 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     1201 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     1261 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     1321 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1381 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1441 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1501 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1561 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1621 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1681 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     1741 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1801 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1861 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1921 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1981 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     2041 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2101 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2161 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     2221 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2281 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2341 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2401 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2461 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2521 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2581 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2641 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2701 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2761 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2821 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2881 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2941 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3001 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3061 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3121 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3181 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3241 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3301 catctgtgcg gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt
     3361 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     3421 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     3481 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     3541 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     3601 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     3661 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     3721 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     3781 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     3841 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     3901 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     3961 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     4021 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     4081 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     4141 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     4201 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     4261 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     4321 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     4381 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     4441 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4501 cgcaattaat gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     4561 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     4621 ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     4681 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     4741 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg
     4801 cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg
     4861 ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca
     4921 ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg
     4981 gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa
     5041 ttatttttga tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg
     5101 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     5161 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat ca
//
