LOCUS       sequence-403601-        6791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of DDX4 low-complexity domain (1-236) with an 
            N-terminal mCherry tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6791)
  AUTHORS   Dumelie JG, Chen Q, Miller D, Attarwala N, Gross SS, Jaffrey SR
  TITLE     Biomolecular condensates create phospholipid-enriched 
            microenvironments.
  JOURNAL   Nat Chem Biol. 2023 Nov 16. doi: 10.1038/s41589-023-01474-4.
  PUBMED    37973889
REFERENCE   2  (bases 1 to 6791)
  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 Chem 
            Biol. 2023 Nov 16. doi: 10.1038/s41589-023-01474-4."
COMMENT     Sequence Label: pET28C-mCherry-DDX4LCD
FEATURES             Location/Qualifiers
     source          1..6791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             21..38
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             42..74
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             81..788
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(226..244)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     494..513
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1550..1567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1620..1638)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1634..1681
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1718..2173
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1805..1824)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2015..2036
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2265..3080)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2265..3080)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2988..3007
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3072..3091
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3202..3790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3691..3710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3944..3961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3976..4115
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4041..4060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4201..4223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4217..4408)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5217..6299)
                     /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     6261..6280
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6300..6377)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6583..6602
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6686..6705
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6686..6704
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6705..6729
                     /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             6744..6766
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(6785..6791,1..11)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatggctagc atgactggtg
       61 gacagcaaat gggtcggatc atggtgagca agggcgagga ggataacatg gccatcatca
      121 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
      181 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
      241 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
      301 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
      361 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
      421 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
      481 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
      541 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
      601 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
      661 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
      721 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
      781 tgtacaaggg tgcgcctggt tctgcaggct ccgcagctgg cggatcgggg atcatgggag
      841 atgaagattg ggaagcagaa atcaaccctc atatgtcttc ctatgttccc atatttgaga
      901 aggataggta ttctggagaa aatggagaca attttaacag gactccagct tcatcatcag
      961 aaatggatga tggaccttct cgaagagatc atttcatgaa aagtggattt gcctctgggc
     1021 ggaattttgg aaacagagat gctggtgagt gtaataagcg agataataca tccacaatgg
     1081 gtggttttgg agttggaaag agttttggaa acagaggttt ttcaaacagc aggtttgaag
     1141 atggtgatag ctctggtttc tggagagagt ctagtaatga ctgcgaagat aatccaacac
     1201 ggaacagagg gttttccaag agaggcggct atcgagatgg aaataattca gaagcttcag
     1261 ggccatacag aagaggtgga agaggtagtt tccgaggttg ccgtggagga tttggtctag
     1321 gaagtccaaa taatgactta gacccagacg aatgtatgca gcgcactggt ggcctttttg
     1381 gttctagaag accagtatta agtggcacag gtaatggtga tacttctcaa agcagaagtg
     1441 gcagtggaag tgaacgaggt ggttacaaag gtttaaatga agaagtaata acaggctctg
     1501 gaaagaattc ttggaagtca gaagcagaag gaggagaaag ttagtcgagc accaccacca
     1561 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1621 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1681 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1741 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1801 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1861 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1921 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1981 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2041 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2101 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2161 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2221 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2281 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2341 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2401 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2461 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2521 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2581 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2641 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2701 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2761 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2821 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2881 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2941 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     3001 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     3061 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3121 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3181 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3241 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3301 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3361 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3421 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3481 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3541 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3601 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3661 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3721 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3781 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3841 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3901 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3961 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4021 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4081 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4141 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4201 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4261 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4321 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4381 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4441 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4501 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4561 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4621 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4681 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4741 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4801 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4861 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4921 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4981 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     5041 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5101 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5161 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5281 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5341 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5401 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5461 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5521 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5581 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5641 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5701 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5761 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5821 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5881 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5941 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     6001 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6061 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6121 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6181 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6241 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6301 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6361 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6421 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     6481 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6541 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     6601 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     6661 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     6721 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcag
     6781 cagccatcat c
//
