LOCUS       sequence-391260-        5634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of recombinant 
            GST-eIF4E K119A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5634)
  AUTHORS   Ogami K, Oishi Y, Hoshino SI
  TITLE     Protocol for analyzing intact mRNA poly(A) tail length using 
            nanopore direct RNA sequencing.
  JOURNAL   STAR Protoc. 2023 May 26;4(2):102340. doi: 
            10.1016/j.xpro.2023.102340.
  PUBMED    37243600
REFERENCE   2  (bases 1 to 5634)
  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: "STAR 
            Protoc."; date: "2023-05-26"; volume: "4(2):102340. doi"; pages: " 
            10.1016/j.xpro.2023.102340"
COMMENT     Sequence Label: pGEX6P-1-eIF4E K119A
FEATURES             Location/Qualifiers
     source          1..5634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1684..1706)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1851..1869)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1937..2041
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2042..2110,2111..2902)
                     /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(2260..2279)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3073..3661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3562..3581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3815..3832
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3905..3982
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3983..5065
                     /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(4002..4021)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5114..5119,5120..5137,5138..5144)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5152..5168
                     /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     5176..5192
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5176..5192
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5188..5634,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5208..5225)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5208..5224)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5217..5239)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc ccggaattca
      961 tggcgactgt cgaaccggaa accaccccta ctcctaatcc cccgactaca gaagaggaga
     1021 aaacggaatc taatcaggag gttgctaacc cagaacacta tattaaacat cccctacaga
     1081 acagatgggc actctggttt tttaaaaatg ataaaagcaa aacttggcaa gcaaacctgc
     1141 ggctgatctc caagtttgat actgttgaag acttttgggc tctgtacaac catatccagt
     1201 tgtctagtaa tttaatgcct ggctgtgact actcactttt taaggatggt attgagccta
     1261 tgtgggaaga tgagaaaaac aaacggggag gacgatggct aattacattg aacgcgcagc
     1321 agagacgaag tgacctcgat cgcttttggc tagagacact tctgtgcctt attggagaat
     1381 cttttgatga ctacagtgat gatgtatgtg gcgctgttgt taatgttaga gctaaaggtg
     1441 ataagatagc aatatggact actgaatgtg aaaacagaga agctgttaca catataggga
     1501 gggtatacaa ggaaaggtta ggacttcctc caaagatagt gattggttat cagtcccacg
     1561 cagacacagc tactaagagc ggctccacca ctaaaaatag gtttgttgtt taagtcgact
     1621 cgagcggccg catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga
     1681 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     1741 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     1801 gacccagtca cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat
     1861 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     1921 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1981 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2041 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2101 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2161 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2221 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2281 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2341 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2401 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2461 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2521 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2581 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2641 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2701 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2761 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2821 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2881 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2941 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3001 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3061 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3121 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3181 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3241 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3301 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3361 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3421 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3481 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3541 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3601 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3661 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3721 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3781 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3841 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa
     3901 ttccgacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     3961 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     4021 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     4081 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     4141 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     4201 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     4261 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4321 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4381 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4441 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     4501 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     4561 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     4621 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     4681 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     4741 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     4801 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     4861 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     4921 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccacgc tagcgcccaa
     4981 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5041 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     5101 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     5161 gataacaatt tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt
     5221 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     5281 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     5341 ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg
     5401 gaaagctggc tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg
     5461 cagatgcacg gttacgatgc gcccatctac accaacgtga cctatcccat tacggtcaat
     5521 ccgccgtttg ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat
     5581 gaaagctggc tacaggaagg ccagacgcga attatttttg atggcgttgg aatt
//
