LOCUS       sequence-464755-        5736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SBP-tagged eIF4A1 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5736)
  AUTHORS   Chen M, Asanuma M, Takahashi M, Shichino Y, Mito M, Fujiwara K, 
            Saito H, Floor SN, Ingolia NT, Sodeoka M, Dodo K, Ito T, Iwasaki S
  TITLE     Dual targeting of DDX3 and eIF4A by the translation inhibitor 
            rocaglamide A.
  JOURNAL   Cell Chem Biol. 2021 Apr 15;28(4):475-486.e8. doi: 
            10.1016/j.chembiol.2020.11.008. Epub 2020 Dec 8.
  PUBMED    33296667
REFERENCE   2  (bases 1 to 5736)
  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.chembiol.2020.11.008"; journalName: "Cell Chem Biol"; 
            date: "2021-04-15- 15"; volume: "28"; issue: "4"; pages: 
            "475-486.e8"
COMMENT     Sequence Label: pColdI-SBP-eIF4A1
FEATURES             Location/Qualifiers
     source          1..5736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..81
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    84..100
                     /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)."
     5'UTR           122..255
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             268..285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             286..396
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     3'UTR           1697..1841
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(1882..1904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2004..2023
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2040..2495)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2177..2198)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2389..2408
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2595..2699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2700..2768,2769..3560)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2918..2937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3731..4319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4220..4239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4491..5573)
                     /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     5535..5554
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5574..5651)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
       61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
      121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
      181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt
      241 aagaggtaat acaccatggg cagcagccat catcatcatc atcacgacga gaaaaccacc
      301 ggctggcggg gaggccacgt ggtggaaggg ctggcaggcg agctggaaca gctgcgggcc
      361 agactggaac accaccccca gggccagaga gagcctagcg gcggaggaat gtctgcgagc
      421 caggattccc gatccagaga caatggcccc gatgggatgg agcccgaagg cgtcatcgag
      481 agtaactgga atgagattgt tgacagcttt gatgacatga acctctcgga gtcccttctc
      541 cgtggcatct acgcctatgg ttttgagaag ccctctgcca tccagcagcg agccattcta
      601 ccttgtatca agggttatga tgtgattgct caagcccaat ctgggactgg gaaaacggcc
      661 acatttgcca tatcgattct gcagcagatt gaattagatc taaaagccac ccaggccttg
      721 gtcctagcac ccactcgaga attggctcag cagatacaga aggtggtcat ggcactagga
      781 gactacatgg gcgcctcctg tcacgcctgt atcgggggca ccaacgtgcg tgctgaggtg
      841 cagaaactgc agatggaagc tccccacatc atcgtgggta cccctggccg tgtgtttgat
      901 atgcttaacc ggagatacct gtcccccaaa tacatcaaga tgtttgtact ggatgaagct
      961 gacgaaatgt taagccgtgg attcaaggac cagatctatg acatattcca aaagctcaac
     1021 agcaacaccc aggtagtttt gctgtcagcc acaatgcctt ctgatgtgct tgaggtgacc
     1081 aagaagttca tgagggaccc cattcggatt cttgtcaaga aggaagagtt gaccctggag
     1141 ggtatccgcc agttctacat caacgtggaa cgagaggagt ggaagctgga cacactatgt
     1201 gacttgtatg aaaccctgac catcacccag gcagtcatct tcatcaacac ccggaggaag
     1261 gtggactggc tcaccgagaa gatgcatgct cgagatttca ctgtatccgc catgcatgga
     1321 gatatggacc aaaaggaacg agacgtgatt atgagggagt ttcgttctgg ctctagcaga
     1381 gttttgatta ccactgacct gctggccaga ggcattgatg tgcagcaggt ttctttagtc
     1441 atcaactatg accttcccac caacagggaa aactatatcc acagaatcgg tcgaggtgga
     1501 cggtttggcc gtaaaggtgt ggctattaac atggtgacag aagaagacaa gaggactctt
     1561 cgagacattg agaccttcta caacacctcc attgaggaaa tgcccctcaa tgttgctgac
     1621 ctcatctgac atatggagct cggtaccctc gagggatccg aattcaagct tgtcgacctg
     1681 cagtctagat aggtaatctc tgcttaaaag cacagaatct aagatccctg ccatttggcg
     1741 gggatttttt tatttgtttt caggaaataa ataatcgatc gcgtaataaa atctattatt
     1801 atttttgtga agaataaatt tgggtgcaat gagaatgcgc aggccctttc gtctcgcgcg
     1861 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     1921 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     1981 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa
     2041 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2101 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat
     2161 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     2221 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccaa
     2281 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     2341 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     2401 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     2461 acccgccgcg cttaatgcgc cgctacaggg cgcgtactat ggttgctttg acgtatgcgg
     2521 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgtca ggtggcactt
     2581 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2641 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2701 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2761 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2821 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2881 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2941 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3001 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3061 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3121 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3181 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3241 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3301 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3361 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3421 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3481 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3541 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3601 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3661 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3721 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3781 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3841 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     3901 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3961 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4021 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4081 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4141 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4201 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4261 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4321 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatag
     4381 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4441 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4561 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4621 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4681 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4741 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4801 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4861 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4921 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4981 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5041 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5101 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5161 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5221 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5281 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5341 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5401 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5461 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5521 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5581 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5641 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5701 cagtagtagg ttgaggccgt tgagcaccgc cgccgc
//
