LOCUS       sequence-290618-        5505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmCup_296-425-Y327AL332A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5505)
  AUTHORS   Igreja C, Peter D, Weiler C, Izaurralde E
  TITLE     4E-BPs require non-canonical 4E-binding motifs and a lateral surface
            of eIF4E to repress translation.
  JOURNAL   Nat Commun. 2014 Sep 2;5:4790. doi: 10.1038/ncomms5790.
  PUBMED    25179781
REFERENCE   2  (bases 1 to 5505)
  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: "Nat 
            Commun."; date: "2014-09-2"; volume: "5:4790. doi"; pages: " 
            10.1038/ncomms5790"
COMMENT     Sequence Label: pGex-CS-DmCup_296-425-Y327AL332A-GB1_Z
FEATURES             Location/Qualifiers
     source          1..5505
                     /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)."
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             906..926
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1555..1577)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1722..1740)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1808..1912
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1913..1981,1982..2773)
                     /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(2131..2150)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2944..3532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3433..3452
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3686..3703
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3776..3853
                     /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             3854..4936
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3873..3892)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4949..4970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4985..4990,4991..5008,5009..5015)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5023..5039
                     /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     5047..5063
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5047..5063
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5059..5505,1..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(5079..5096)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5079..5095)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5088..5110)
                     /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 atctcgagaa tctttatttt cagggcgcca tgggcagcac aggcattcac aagccaggaa
      961 gtctgcgagc acccaaggct gttcgtccta ctactgctcc ggtggtcagc agcaagccgg
     1021 tcaagagcgc tacccgctcc cgcgcgatgg acattcgcaa cgggatgttc aatgccctga
     1081 tgcacaggtc caaggagagc tttgtgatgc ctcgcatcgc cacctgcgac gacattgagt
     1141 tggagggaag gcttcgccgc atgaatattt ggcgcacctc ggacggcaca agattccgga
     1201 cccgctccac cacagctaac ttaaacatga acaacaacaa caacaatgag tgcatgcctg
     1261 ccttcttcaa aaacaaaaat aagccgaacc taatcagtga cgagtccatc atccagagcc
     1321 aaccgatgca gtacaaactt atcctgaacg gtaaaaccct gaaaggtgaa accaccaccg
     1381 aagctgttga cgctgctacc gcggaaaaag ttttcaaaca gtacgctaac gacaacggtg
     1441 ttgacggtga atggacctac gacgacgcta ccaaaaccta caccgtaacg gaataagaat
     1501 tcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     1561 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     1621 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     1681 acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga tacgcctatt
     1741 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     1801 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1861 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1921 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1981 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2041 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2101 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     2161 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2221 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2281 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2341 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2401 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     2461 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2521 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2581 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2641 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2701 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2761 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2821 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2881 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2941 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3001 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3061 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3121 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3181 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3241 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3301 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3361 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3421 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3481 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3541 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3601 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3661 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3721 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa attccgacac
     3781 catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     3841 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     3901 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     3961 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     4021 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     4081 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     4141 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     4201 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     4261 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     4321 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     4381 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     4441 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     4501 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     4561 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     4621 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     4681 cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt ttcgcctgct
     4741 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     4801 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     4861 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     4921 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     4981 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     5041 ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt tttacaacgt
     5101 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5161 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5221 ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg
     5281 ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac
     5341 ggttacgatg cgcccatcta caccaacgta acctatccca ttacggtcaa tccgccgttt
     5401 gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga tgaaagctgg
     5461 ctacaggaag gccagacgcg aattattttt gatggcgttg gaatt
//
