LOCUS       sequence-315912-        7374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of EIF4E_IF4E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7374)
  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 7374)
  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_EIF4E_IF4E
FEATURES             Location/Qualifiers
     source          1..7374
                     /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      1569..1616
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3209..3664)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3346..3367)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3558..3577
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3706..3728)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3814..3954
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3869..3888
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3969..3986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4140..4727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4898..5689,5690..5758))
                     /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     5521..5540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5759..5863)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5931..5949
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5972..6049
                     /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             6050..7132
                     /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(6069..6088)
                     /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 cggccgctac cgtcgaaccg gaaaccacgc cgacgccgaa tccgccgacc acggaagaag
      961 aaaaaaccga aagcaaccag gaagttgcga atccggaaca ttatattaaa cacccgctgc
     1021 aaaaccgttg ggccctgtgg tttttcaaaa acgataaaag caaaacctgg caggcaaacc
     1081 tgcgcctgat ttctaaattt gatacggtgg aagacttctg ggccctgtat aaccatatcc
     1141 aactgagctc taatctgatg ccgggttgcg attacagtct gtttaaagac ggcattgaac
     1201 cgatgtggga agatgagaaa aacaaacgtg gcggtcgctg gctgatcacc ctgaacaaac
     1261 agcaacgtcg ctcagatctg gaccgttttt ggctggaaac cctgctgtgc ctgattggtg
     1321 aatcattcga tgactactcg gatgacgtgt gtggtgcagt ggttaatgtt cgtgctaaag
     1381 gcgataaaat tgcgatctgg accacggaat gtgaaaaccg cgaagcggtt acccatatcg
     1441 gccgtgtcta taaagaacgc ctgggtctgc cgccgaaaat tgttatcggt taccagtccc
     1501 acgcagacac cgcaacgaaa agtggctcca ccacgaaaaa ccgcttcgtc gtggcggccg
     1561 cataataaac cgatacaatt aaaggctcct tttggagcct tttttttgga gattttcaac
     1621 gtgaaaaaat tattattcgc aattccttta gttgttcctt tctattctca ctccgctgaa
     1681 actgttgaaa gttgtttagc aaaaccccat acagaaaatt catttactaa cgtctggaaa
     1741 gacgacaaaa ctttagatcg ttacgctaac tatgagggct gtctgtggaa tgctacaggc
     1801 gttgtagttt gtactggtga cgaaactcag tgtctagcta gagtggcggt ggctctggtt
     1861 ccggtgattt tgattatgaa aagatggcaa acgctaataa gggggctatg accgaaaatg
     1921 ccgatgaaaa cgcgctacag tctgacgcta aaggcaaact tgattctgtc gctactgatt
     1981 acggtgctgc tatcgatggt ttcattggtg acgtttccgg ccttgctaat ggtaatggtg
     2041 ctactggtga ttttgctggc tctaattccc aaatggctca agtcggtgac ggtgataatt
     2101 cacctttaat gaataatttc cgtcaatatt taccttccct ccctcaatcg gttgaatgtc
     2161 gcccttttgt ctttggcgct ggtaaaccat atgaattttc tattgattgt gacaaaataa
     2221 acttattccg tggtgtcttt gcgtttcttt tatatgttgc cacctttatg tatgtatttt
     2281 ctacgtttgc taacatactg cgtaataagg agtcttaatc atgccagttc ttttggctag
     2341 cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac
     2401 ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga
     2461 gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc
     2521 atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg
     2581 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     2641 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     2701 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     2761 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     2821 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     2881 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     2941 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     3001 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     3061 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     3121 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     3181 gctgatgagc tttaccgcag gatccgcgaa attgtaagcg ttaatatttt gttaaaattc
     3241 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     3301 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     3361 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3421 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     3481 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     3541 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3601 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3661 gcgtcgcgga tcctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3721 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     3781 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     3841 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     3901 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     3961 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4021 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4081 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4141 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4201 cgaaacccga caggactata aagataccag ggtttccccc tggaagctcc ctcgtgcgct
     4261 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4321 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4381 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4441 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4501 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4561 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4621 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4681 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4741 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4801 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4861 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4921 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4981 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5041 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5101 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5161 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     5221 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5281 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5341 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5401 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5461 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg
     5521 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5581 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5641 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5701 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5761 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5821 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5881 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     5941 tcacgaggcc ctttcgtctt caagaattcc cgacaccatc gaatggtgca aaacctttcg
     6001 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt
     6061 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     6121 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     6181 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     6241 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     6301 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     6361 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     6421 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     6481 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     6541 agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     6601 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     6661 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     6721 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     6781 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     6841 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
     6901 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     6961 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
     7021 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7081 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7141 caattaatgt aagttagctc actcattagg cacaattctc atgtttgaca gcttatcatc
     7201 gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct
     7261 gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata
     7321 atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctg
//
