LOCUS       sequence-356583-        7025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmThor_50-83-R56ER63E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7025)
  AUTHORS   Peter D, Igreja C, Weber R, Wohlbold L, Weiler C, Ebertsch L, 
            Weichenrieder O, Izaurralde E
  TITLE     Molecular architecture of 4E-BP translational inhibitors bound to 
            eIF4E.
  JOURNAL   Mol Cell. 2015 Mar 19;57(6):1074-87. doi: 
            10.1016/j.molcel.2015.01.017. Epub 2015 Feb 19.
  PUBMED    25702871
REFERENCE   2  (bases 1 to 7025)
  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.2015.01.017"; journalName: "Mol Cell"; date: 
            "2015-03-19- 19"; volume: "57"; issue: "6"; pages: "1074-87"
COMMENT     Sequence Label: pnEA-NpM-DmThor_50-83-R56ER63E-GB1_AA
FEATURES             Location/Qualifiers
     source          1..7025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             88..1188
                     /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             1204..1227
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1527..1545)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1541..1588
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1763..1768,1769..1785,1786..1791))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1818..1836)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1904..2008
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2009..2077,2078..2869)
                     /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(2227..2246)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3040..3628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3529..3548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3782..3799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3814..3953
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3879..3898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4039..4061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4055..4246)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6601..6620
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6640..6717)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6923..6942
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttccccacta gtaataattt
       61 tgtttaactt taagaaggag atataccatg gaaactgaag aaggtaaact ggtaatctgg
      121 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
      181 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
      241 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
      301 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
      361 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
      421 gaagcgttat cgctgattta taacaaagac ctgctgccga acccgccaaa aacctgggaa
      481 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgccct gatgttcaac
      541 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
      601 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
      661 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
      721 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
      781 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
      841 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
      901 ccgaacaaag agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
      961 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     1021 ttggcgaaag atccacgtat tgccgccacg atggaaaacg cccagaaagg tgaaatcatg
     1081 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     1141 gccagcggtc gtcagactgt cgatgaagcc cttaaagacg cgcagactgg taccggcagc
     1201 ggcctggaag ttctgttcca ggggccccat atgaccaaac ttatctacga ggaggctttc
     1261 atgaagaatc tcgagggctc cccattgagc caaactccgc cgtccaacgt gcccagttgc
     1321 ttgctgaggg gaactgctag catgcagtac aaacttatcc tgaacggtaa aaccctgaaa
     1381 ggtgaaacca ccaccgaagc tgttgacgct gctaccgcgg aaaaagtttt caaacagtac
     1441 gctaacgaca acggtgttga cggtgaatgg acctacgacg acgctaccaa aacctacacc
     1501 gtaacggaat gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1561 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatatcccgc
     1621 aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg
     1681 ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa
     1741 tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa
     1801 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     1861 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1921 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1981 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2041 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2101 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     2161 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2221 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     2281 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2341 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2401 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2461 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2521 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     2581 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2641 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2701 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2761 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2821 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2881 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     2941 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3001 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3061 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3181 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3421 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3661 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3721 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3781 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3841 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3901 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3961 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4021 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4081 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4141 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4201 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4261 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4321 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4381 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4441 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4501 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4561 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4621 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4681 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     4741 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     4801 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     4861 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     4921 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     4981 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     5041 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     5101 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     5161 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     5221 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcag gaccagtgac
     5281 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5341 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5401 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5461 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5521 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5581 aaactgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5641 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5701 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5761 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5821 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5881 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5941 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6001 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6061 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6121 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6181 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6241 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6301 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6361 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6421 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6481 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6541 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6601 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6661 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6721 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6781 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6841 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6901 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6961 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     7021 gaaat
//
