LOCUS       sequence-232424-        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(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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1080..1102)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1247..1265)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1333..1437
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1438..1506,1507..2298)
                     /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(1656..1675)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2469..3057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2958..2977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3211..3228
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3301..3378
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3379..4461
                     /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(3398..3417)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4510..4515,4516..4533,4534..4540)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4548..4564
                     /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     4572..4588
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4572..4588
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4584..5111
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4604..4621)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4604..4620)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4613..4635)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tccccttcta agacaagcaa cactatccgt
      781 gttttcttgc cgaacaagca aagaacagtg gtcaatgtgc gaaatggaat gagcttgcat
      841 gactgcctta tgaaagcact caaggtgagg ggcctgcaac cagagtgctg tgcagtgttc
      901 agacttctcc acgaacacaa aggtaaaaaa gcacgcttag attggaatac tgatgctgcg
      961 tctttgattg gagaagaact tcaagtagat ttcctgtagg aattcccggg tcgactcgag
     1021 cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     1081 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1141 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     1201 cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc
     1261 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     1321 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1381 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1441 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1501 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1561 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1621 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1681 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1741 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1801 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1861 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1921 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1981 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2041 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2101 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2161 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2221 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2281 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2341 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2401 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2461 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2521 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2581 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2641 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2701 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2761 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2821 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2881 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2941 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3001 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3061 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3121 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3181 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3241 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc
     3301 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     3361 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     3421 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     3481 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     3541 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     3601 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     3661 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     3721 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     3781 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     3841 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     3901 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     3961 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     4021 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     4081 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     4141 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     4201 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     4261 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     4321 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     4381 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4441 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     4501 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     4561 acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac
     4621 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4681 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4741 gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa
     4801 gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga
     4861 tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc
     4921 cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa
     4981 gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt agcttatcga
     5041 ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt
     5101 gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat
     5161 gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tg
//
