LOCUS       sequence-217169-        6285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally GST-tagged mouse Eif4e K119A in bacterial 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6285)
  AUTHORS   Trotman JB, Giltmier AJ, Mukherjee C, Schoenberg DR
  TITLE     RNA guanine-7 methyltransferase catalyzes the methylation of 
            cytoplasmically recapped RNAs.
  JOURNAL   Nucleic Acids Res. 2017 Oct 13;45(18):10726-10739. doi: 
            10.1093/nar/gkx801.
  PUBMED    28981715
REFERENCE   2  (bases 1 to 6285)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2017-10-13- 13"; volume: "45"; issue: 
            "18"; pages: "10726-10739"
COMMENT     Sequence Label: pGEX-2T-GST-eIF4E K119A
FEATURES             Location/Qualifiers
     source          1..6285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(72..77,78..93,94..100)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    108..124
                     /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             147..800
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDQMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     758..780
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             807..824
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2224..2246)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2391..2409)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2477..2581
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2582..2650,2651..3442)
                     /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(2800..2819)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3613..4201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4102..4121
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4355..4372
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4445..4522
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4523..5605
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLAMHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4542..4561)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5654..5659,5660..5677,5678..5684)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5692..5708
                     /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     5716..5732
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5716..5732
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5728..6255
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5748..5765)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5748..5764)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5757..5779)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
       61 gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
      121 acaatttcac acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg
      181 gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt
      241 tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt
      301 ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct atggccatca
      361 tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga
      421 tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata
      481 gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa
      541 tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg
      601 acttcatgtt gtatgacgct cttgatgttg ttttatacat ggaccaaatg tgcctggatg
      661 cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt
      721 acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg
      781 gtggcgacca tcctccaaaa tcggatctgg ttccgcgtgg atcccccatg gcgactgtgg
      841 aaccggaaac cacccctacc actaatcccc cacctgcaga agaggaaaaa acagagtcta
      901 atcaggaggt tgctaaccca gagcactata ttaaacaccc tctacagaac aggtgggcac
      961 tctggttttt taaaaatgat aaaagcaaaa cttggcaagc aaaccttcga ttgatctcta
     1021 agtttgatac tgttgaagac ttttgggctc tatacaacca tatccagttg tctagtaatt
     1081 taatgcctgg ctgtgactac tcacttttta aggacgggat tgagcctatg tgggaagatg
     1141 agaaaaacaa acgaggagga cggtggctga tcacactgaa cgcgcagcag agacggagtg
     1201 acctcgatcg cttctggcta gagacactgc tgtgccttat tggagaatct ttcgatgact
     1261 acagtgatga tgtgtgtgga gctgttgtta atgttagagc taaaggtgat aagatagcaa
     1321 tatggactac tgagtgtgaa aacagagatg cagtcacaca catagggagg gtatacaagg
     1381 aaaggttagg acttcctccg aagatagtga ttggttatca gtcccacgca gacacagcta
     1441 caaagagcgg ctccaccact aaaaataggt ttgttgttta agaagacacc ttctgagtat
     1501 tctcacagga gactgcgtca cgcaatcgag attgggagct gaaccaaagc ctcatcaaag
     1561 cagagtggac tgcactgaag ttgattccat ccaagtgttg ctaagatata agagaagtct
     1621 cattcgcctt tgtcttgtac ttctgtgttc attctcctcc cccaccccca atttttgcta
     1681 gtgtgtccac tatcccaatc aaagaattac agtatacgtc acccaagaac ccgcagatgt
     1741 gttcctggcc cgctctgtaa cagccggtta gaattaccat gacacacaca tttgcctttc
     1801 cacagtattc gaaaaagaac ttgcatttct attaccttag caggaaagat ctggttttgc
     1861 tccactccat gcaggagcgg actttgctgg tgtgagagtc tgagtacagc tttctagcaa
     1921 ccttctgttt cctttcacag cattgtcctt gctgtcctct tgctgatggc tgctagattt
     1981 aatttatttg cttccatcct tgataacatt agtgattctg atttcagttt ttcatttgtt
     2041 ttgcttttgt ttttttcctc gtgtaacatt ggtgaaggat ccaggaatat gacagaaagg
     2101 tggaataaac attaaatttg tgcattcttt ggtaattttt ttgttttttg taactacaaa
     2161 gctgggaatt catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga
     2221 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     2281 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     2341 gacccagtca cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat
     2401 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggaac
     2461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2521 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2581 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2641 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2701 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2761 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2821 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2881 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2941 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3001 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3061 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3121 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3181 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3241 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3301 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3361 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3421 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3481 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3541 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3601 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3661 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3721 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3781 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3841 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3901 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3961 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4021 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4081 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4141 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4201 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4261 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4321 tgataccgct cgccgcagcc gaacgacaga gcgcagcgag tcagtgagcg aggaagcgga
     4381 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa
     4441 ttccgacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     4501 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     4561 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     4621 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     4681 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccat
     4741 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     4801 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4861 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4921 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4981 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     5041 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     5101 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     5161 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     5221 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     5281 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     5341 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     5401 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     5461 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     5521 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5581 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     5641 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     5701 gataacaatt tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt
     5761 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     5821 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     5881 ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg
     5941 gaaagctggc tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg
     6001 cagatgcacg gttacgatgc gcccatctac accaacgtga cctatcccat tacggtcaat
     6061 ccgccgtttg ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat
     6121 gaaagctggc tacaggaagg ccagacgcga attatttttg atggcgttgg aattagctta
     6181 tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg
     6241 ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcg
//
