LOCUS       sequence-291176-        4970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of SceIF4E_25-213-K42AK168AK187A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4970)
  AUTHORS   Gruner S, Weber R, Peter D, Chung MY, Igreja C, Valkov E, Izaurralde
            E
  TITLE     Structural motifs in eIF4G and 4E-BPs modulate their binding to 
            eIF4E to regulate translation initiation in yeast.
  JOURNAL   Nucleic Acids Res. 2018 Jul 27;46(13):6893-6908. doi: 
            10.1093/nar/gky542.
  PUBMED    30053226
REFERENCE   2  (bases 1 to 4970)
  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: "2018-07-27- 27"; volume: "46"; issue: 
            "13"; pages: "6893-6908"
COMMENT     Sequence Label: pnYC-NpH-SceIF4E_25-213-K42AK168AK187A_AF
FEATURES             Location/Qualifiers
     source          1..4970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2294..2313
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2402..2421
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2402..2420
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2421..2445
                     /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)."
     RBS             2460..2482
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2501..2518
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2540..2563
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3202..3220)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3216..3263
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3438..3443,3444..3460,3461..3466))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      3578..4123
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     4024..4043
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4240..4257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4649..4751
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(4752..4970,1..441)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFGMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4818..4837)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 ggtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
       61 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
      121 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
      181 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
      241 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
      301 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
      361 gccgtctgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
      421 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
      481 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga
      541 cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac
      601 cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt
      661 tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca
      721 gagcctgata aaaacggtta gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      781 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgcttg tttcggcgtg
      841 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ctgccggcac ctgtcctacg
      901 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      961 cggaaggagc taccggacag cggtgcggac tgttgtaact cagaataaga aatgaggccg
     1021 ctcatggcgt tgactctcag tcatagtatc gtggtatcac cggttggttc cactctctgt
     1081 tgcgggcaac ttcagcagca cgtaggggac ttccgcgttt ccagacttta cgaaacacgg
     1141 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     1201 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     1261 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     1321 cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa
     1381 gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg
     1441 gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
     1501 caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca
     1561 acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccagtga
     1621 tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat
     1681 ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa
     1741 gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca
     1801 gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     1861 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     1921 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     1981 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     2041 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac
     2101 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     2161 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     2221 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     2281 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     2341 tgatgccggc cacgatgcgt ccggcgtaga ggatcagact aagatctcga tcccgcgaaa
     2401 ttaatacgac tcactatagg ggaattgtga gcggataaca attccccact agtaataatt
     2461 ttgtttaact ttaagaagga gatataccat gggcagcagc catcatcatc atcatcacag
     2521 cagcggtacc ggcagcggcc tggaagttct gttccagggg ccccatatga ccgttctgag
     2581 cgatagcgca cattttgatg ttaaacatcc gctgaatacc gcgtggaccc tgtggtatac
     2641 aaaaccggca gttgataaaa gcgaaagctg gtcagatctg ctgcgtccgg ttaccagttt
     2701 tcagaccgtt gaagaatttt gggccattat tcagaatatt ccggaaccgc atgaactgcc
     2761 gctgaaaagc gattatcatg tgtttcgtaa tgatgttcgt ccggaatggg aagatgaagc
     2821 aaatgcaaaa ggtggcaaat ggtcatttca gctgcgtggt aaaggtgcag atattgatga
     2881 actgtggctg cgtaccctgc tggcagttat tggtgaaacc attgatgaag atgatagcca
     2941 gattaatggt gtggttctga gcattcgtaa aggcggtaac aaatttgcac tgtggaccgc
     3001 gagcgaagat aaagaaccgc tgctgcgtat tggtggtaaa ttcaaacagg ttctggcgct
     3061 gaccgatgat ggtcacctgg aattttttcc gcatagcagc gccaatggtc gtcatcctca
     3121 accatcaatc accttgtaag ctagccctag gagatccggc tgctaacaaa gcccgaaagg
     3181 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3241 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
     3301 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     3361 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     3421 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattaca
     3481 acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat tgcactgaaa
     3541 tcaagaaata ttttatctga ttaataagat gatcttcttg agatcgtttt ggtctgcgcg
     3601 taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct
     3661 gagctaccaa ctctttgaac cgaggtaact ggcttggagg agcgcagtca ccaaaacttg
     3721 tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta
     3781 ccagtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag
     3841 ttaccggata aggcgcagcg gtcggactga acggggggtt cgtgcataca gtccagcttg
     3901 gagcgaactg cctacccgga actgagtgtc aggcgtggaa tgagacaaac gcggccataa
     3961 cagcggaatg acaccggtaa accgaaaggc aggaacagga gagcgcacga gggagccgcc
     4021 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccaccact gatttgagcg
     4081 tcagatttcg tgatgcttgt caggggggcg gagcctatgg aaaaacggct ttgccgcggc
     4141 cctctcactt ccctgttaag tatcttcctg gcatcttcca ggaaatctcc gccccgttcg
     4201 taagccattt ccgctcgccg cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa
     4261 gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg
     4321 ccacatgaag cacttcactg acaccctcat cagtgccaac atagtaagcc agtatacact
     4381 cccctagcgc tgatgtccgg cggtgctttt gccgttacgc accaccccgt cagtagctga
     4441 acaggaggga cagctgatag aaacagaagc cactggagca cctcaaaaac accatcatac
     4501 actaaatcag taagttggca gcatcacccg acgcactttg cgccgaataa atacctgtga
     4561 cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc gggaagccct
     4621 gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc aactttcacc
     4681 ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta
     4741 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     4801 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     4861 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     4921 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc
//
