LOCUS       sequence-381357-        7110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of 
            DmeIF4Ga_617-630-DmThor_64-71-DmeIF4Ga_639-676.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7110)
  AUTHORS   Gruner S, Peter D, Weber R, Wohlbold L, Chung MY, Weichenrieder O, 
            Valkov E, Igreja C, Izaurralde E
  TITLE     The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface
            to Regulate Translation Initiation.
  JOURNAL   Mol Cell. 2016 Nov 3;64(3):467-479. doi: 
            10.1016/j.molcel.2016.09.020. Epub 2016 Oct 20.
  PUBMED    27773676
REFERENCE   2  (bases 1 to 7110)
  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: 
            "10.1016/j.molcel.2016.09.020"; journalName: "Mol Cell"; date: 
            "2016-11-3- 3"; volume: "64"; issue: "3"; pages: "467-479"
COMMENT     Sequence Label: 
            pnEA-NpM-DmeIF4Ga_617-630-DmThor_64-71-DmeIF4Ga_639-676-GB1_AD
FEATURES             Location/Qualifiers
     source          1..7110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2079..2098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2239..2261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2255..2446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4801..4820
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4840..4917)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5123..5142
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5226..5245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5226..5244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5245..5269
                     /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             5284..5306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5313..6413
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="METEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     CDS             6429..6452
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(6837..6855)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6851..6898
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7073..7078,7079..7095,7096..7101))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2101 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2281 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2341 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2401 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2461 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2521 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2581 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2641 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     2701 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     2761 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     2821 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     2881 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     2941 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     3001 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     3061 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     3121 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     3181 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     3241 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     3301 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     3361 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     3421 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3481 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3541 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3601 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3661 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3721 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3781 aaactgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3841 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3901 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3961 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4021 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4081 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4141 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4201 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4261 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4321 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4381 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4441 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4501 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4561 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4621 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4681 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4741 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4801 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4861 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4921 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4981 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5041 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5101 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5161 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     5221 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc cactagtaat
     5281 aattttgttt aactttaaga aggagatata ccatggaaac tgaagaaggt aaactggtaa
     5341 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
     5401 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
     5461 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
     5521 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
     5581 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
     5641 ctgttgaagc gttatcgctg atttataaca aagacctgct gccgaacccg ccaaaaacct
     5701 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gccctgatgt
     5761 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
     5821 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
     5881 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
     5941 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
     6001 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
     6061 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg
     6121 ccagtccgaa caaagagctg gcgaaagagt tcctcgaaaa ctatctgctg actgatgaag
     6181 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     6241 aagagttggc gaaagatcca cgtattgccg ccacgatgga aaacgcccag aaaggtgaaa
     6301 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     6361 acgccgccag cggtcgtcag actgtcgatg aagcccttaa agacgcgcag actggtaccg
     6421 gcagcggcct ggaagttctg ttccaggggc cccatatggg aaaaaaacaa tacgaccgag
     6481 aacagttatt acagttacgc ggctccccat tgagccaaac tccgccagag gtaaaaaatg
     6541 tgtctattct tcctcaacca aacttaatgc catcatttat tcgaaataac aataataaca
     6601 agcgagtaca atcgatggtt ggtattattg gcaatggatc cgctagcggc atgcagtaca
     6661 aacttatcct gaacggtaaa accctgaaag gtgaaaccac caccgaagct gttgacgctg
     6721 ctaccgcgga aaaagttttc aaacagtacg ctaacgacaa cggtgttgac ggtgaatgga
     6781 cctacgacga cgctaccaaa acctacaccg taacggaatg agttggctgc tgccacccgc
     6841 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     6901 gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag
     6961 cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat
     7021 ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc
     7081 ttatcgatga taagctgtca aacatgagaa
//
