LOCUS       sequence-465604-        5946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SBP-tagged helicase core region of DDX3X in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5946)
  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 5946)
  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-DDX3_helicase_core
FEATURES             Location/Qualifiers
     source          1..5946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..80
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    83..99
                     /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           121..254
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             267..284
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             285..395
                     /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           1906..2050
                     /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(2091..2113)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2213..2232
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2249..2704)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2386..2407)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2598..2617
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2804..2908
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2909..2977,2978..3769)
                     /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(3127..3146)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3940..4528
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4429..4448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4700..5782)
                     /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     5744..5763
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5783..5860)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggaatggtg tggccgatta atcataaata tgaaaaataa ttgttgcatc acccgccaat
       61 gcgtggctta atgcacatca aattgtgagc ggataacaat ttgatgtgct agcgcatatc
      121 cagtgtagta aggcaagtcc cttcaagagt tatcgttgat acccctcgta gtgcacattc
      181 ctttaacgct tcaaaatctg taaagcacgc catatcgccg aaaggcacac ttaattatta
      241 agaggtaata caccatgggc agcagccatc atcatcatca tcacgacgag aaaaccaccg
      301 gctggcgggg aggccacgtg gtggaagggc tggcaggcga gctggaacag ctgcgggcca
      361 gactggaaca ccacccccag ggccagagag agcctagcgg cggaggagat gaagatgatt
      421 ggtcaaaacc actcccacca agtgaacgct tggaacagga actcttttct ggaggcaaca
      481 ctgggattaa ttttgagaaa tacgatgaca ttccagttga ggcaacaggc aacaactgtc
      541 ctccacatat tgaaagtttc agtgatgttg agatgggaga aattatcatg ggaaacattg
      601 agcttactcg ttatactcgc ccaactccag tgcaaaagca tgctattcct attatcaaag
      661 agaaaagaga cttgatggct tgtgcccaaa cagggtctgg aaaaactgca gcatttctgt
      721 tgcccatctt gagtcagatt tattcagatg gtccaggcga ggctttgagg gccatgaagg
      781 aaaatggaag gtatgggcgc cgcaaacaat acccaatctc cttggtatta gcaccaacga
      841 gagagttggc agtacagatc tacgaggaag ccagaaaatt ttcataccga tctagagttc
      901 gtccttgcgt ggtttatggt ggtgccgata ttggtcagca gattcgagac ttggaacgtg
      961 gatgccattt gttagtagcc actccaggac gtctagtgga tatgatggaa agaggaaaga
     1021 ttggattaga cttttgcaaa tacttggtgt tagatgaagc tgatcggatg ttggatatgg
     1081 ggtttgagcc tcagattcgt agaatagtcg aacaagatac tatgcctcca aagggtgtcc
     1141 gccacactat gatgtttagt gctacttttc ctaaggaaat acagatgctg gctcgtgatt
     1201 tcttagatga atatatcttc ttggctgtag gaagagttgg ctctacctct gaaaacatca
     1261 cacagaaagt agtttgggtg gaagaatcag acaaacggtc atttctgctt gacctcctaa
     1321 atgcaacagg caaggattca ctgaccttag tgtttgtgga gaccaaaaag ggtgcagatt
     1381 ctctggagga tttcttatac catgaaggat acgcatgtac cagcatccat ggagaccgtt
     1441 ctcagaggga tagagaagag gcccttcacc agttccgctc aggaaaaagc ccaattttag
     1501 tggctacagc agtagcagca agaggactgg acatttcaaa tgtgaaacat gttatcaatt
     1561 ttgacttgcc aagtgatatt gaagaatatg tacatcgtat tggtcgtacg ggacgtgtag
     1621 gaaaccttgg cctggcaacc tcattcttta acgagaggaa cataaatatt actaaggatt
     1681 tgttggatct tcttgttgaa gctaaacaag aagtgccgtc ttggttagaa aacatggctt
     1741 atgaacacca ctacaagggt agcagtcgtg gacgttctaa gagtagcaga tttagtggag
     1801 ggtttggtgc cagagactac cgacaaagta gcggttgaca tatggagctc ggtaccctcg
     1861 agggatccga attcaagctt gtcgacctgc agtctagata ggtaatctct gcttaaaagc
     1921 acagaatcta agatccctgc catttggcgg ggattttttt atttgttttc aggaaataaa
     1981 taatcgatcg cgtaataaaa tctattatta tttttgtgaa gaataaattt gggtgcaatg
     2041 agaatgcgca ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2101 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2161 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     2221 gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt gttaaaattc
     2281 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2341 ccttataaat caaaagaata gcccgagata gggttgagtg ttgttccagt ttggaacaag
     2401 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2461 gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa
     2521 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2581 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2641 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2701 gcgtactatg gttgctttga cgtatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     2761 aataccgcat caggcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2821 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2881 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2941 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3001 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3061 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3121 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3181 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3241 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3301 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3361 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3421 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3481 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3541 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3601 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3661 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3721 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3781 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3841 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3901 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3961 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4021 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4081 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4141 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4201 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4261 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4321 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4381 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4441 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4501 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4561 ggccttttgc tggccttttg ctcacatagt catgccccgc gcccaccgga aggagctgac
     4621 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4681 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4801 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4861 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4921 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4981 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5041 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5101 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5161 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5221 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5281 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5341 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5401 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5461 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5521 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5581 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5641 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5701 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5761 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5821 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5881 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5941 gccgct
//
