LOCUS       sequence-464754-        5805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His-tagged helicase core region of DDX3X with Q360P 
            mutation in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5805)
  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 5805)
  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-DDX3_helicase_core_Q360P
FEATURES             Location/Qualifiers
     source          1..5805
                     /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             256..270
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             289..300
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           1766..1910
                     /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(1951..1973)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2073..2092
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2109..2564)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2246..2267)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2458..2477
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2664..2768
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2769..2837,2838..3629)
                     /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(2987..3006)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3800..4388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4289..4308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4560..5642)
                     /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     5604..5623
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5643..5720)
                     /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 acaccatgaa tcacaaagtg catcatcatc atcatcatat cgaaggtagg
      301 catgatgaag atgattggtc aaaaccactc ccaccaagtg aacgcttgga acaggaactc
      361 ttttctggag gcaacactgg gattaatttt gagaaatacg atgacattcc agttgaggca
      421 acaggcaaca actgtcctcc acatattgaa agtttcagtg atgttgagat gggagaaatt
      481 atcatgggaa acattgagct tactcgttat actcgcccaa ctccagtgca aaagcatgct
      541 attcctatta tcaaagagaa aagagacttg atggcttgtg cccaaacagg gtctggaaaa
      601 actgcagcat ttctgttgcc catcttgagt cagatttatt cagatggtcc aggcgaggct
      661 ttgagggcca tgaaggaaaa tggaaggtat gggcgccgca aacaataccc aatctccttg
      721 gtattagcac caacgagaga gttggcagta cagatctacg aggaagccag aaaattttca
      781 taccgatcta gagttcgtcc ttgcgtggtt tatggtggtg ccgatattgg tcagcagatt
      841 cgagacttgg aacgtggatg ccatttgtta gtagccactc caggacgtct agtggatatg
      901 atggaaagag gaaagattgg attagacttt tgcaaatact tggtgttaga tgaagctgat
      961 cggatgttgg atatggggtt tgagcctccg attcgtagaa tagtcgaaca agatactatg
     1021 cctccaaagg gtgtccgcca cactatgatg tttagtgcta cttttcctaa ggaaatacag
     1081 atgctggctc gtgatttctt agatgaatat atcttcttgg ctgtaggaag agttggctct
     1141 acctctgaaa acatcacaca gaaagtagtt tgggtggaag aatcagacaa acggtcattt
     1201 ctgcttgacc tcctaaatgc aacaggcaag gattcactga ccttagtgtt tgtggagacc
     1261 aaaaagggtg cagattctct ggaggatttc ttataccatg aaggatacgc atgtaccagc
     1321 atccatggag accgttctca gagggataga gaagaggccc ttcaccagtt ccgctcagga
     1381 aaaagcccaa ttttagtggc tacagcagta gcagcaagag gactggacat ttcaaatgtg
     1441 aaacatgtta tcaattttga cttgccaagt gatattgaag aatatgtaca tcgtattggt
     1501 cgtacgggac gtgtaggaaa ccttggcctg gcaacctcat tctttaacga gaggaacata
     1561 aatattacta aggatttgtt ggatcttctt gttgaagcta aacaagaagt gccgtcttgg
     1621 ttagaaaaca tggcttatga acaccactac aagggtagca gtcgtggacg ttctaagagt
     1681 agcagattta gtggagggtt tggtgccaga gactaccgac aaagtagcgg ttgaaagctt
     1741 gtcgacctgc agtctagata ggtaatctct gcttaaaagc acagaatcta agatccctgc
     1801 catttggcgg ggattttttt atttgttttc aggaaataaa taatcgatcg cgtaataaaa
     1861 tctattatta tttttgtgaa gaataaattt gggtgcaatg agaatgcgca ggccctttcg
     1921 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     1981 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     2041 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     2101 gcaccataaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     2161 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     2221 gcccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2281 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2341 atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2401 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2461 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2521 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtactatg gttgctttga
     2581 cgtatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgtcag
     2641 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2701 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2761 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2821 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2881 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2941 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3001 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3061 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3121 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3181 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3241 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3301 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3361 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3421 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3481 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3541 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3601 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3661 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3721 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3781 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3841 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3901 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3961 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4021 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4081 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4141 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4201 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4261 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4321 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4381 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4441 ctcacatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4501 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4561 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4621 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4681 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4741 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     4801 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     4861 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     4921 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     4981 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5041 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5101 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5161 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5221 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5281 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5341 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5401 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5461 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5521 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5581 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5641 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5701 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     5761 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgc
//
