LOCUS       sequence-403599-        7604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of MED1 low-complexity domain (947-1581) with an 
            N-terminal mCherry tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7604)
  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 7604)
  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-MED1LCD
FEATURES             Location/Qualifiers
     source          1..7604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..41)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     232..253
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(482..1297)
                     /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(482..1297)
                     /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     1205..1224
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1289..1308
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1419..2007
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1908..1927
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2161..2178
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2193..2332
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2258..2277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2418..2440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2434..2625)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3434..4516)
                     /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     4478..4497
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4517..4594)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4800..4819
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4903..4922
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4903..4921
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4922..4946
                     /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             4961..4983
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5002..5019
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5029..5046
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5050..5082
                     /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             5089..5796
                     /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(5234..5252)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5502..5521
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             7371..7388
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7441..7459)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7455..7502
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(7539..7604,1..390)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
       61 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      121 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      181 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      241 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      301 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      361 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
      421 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      481 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      541 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      601 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      661 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      721 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
      781 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
      841 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
      901 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
      961 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1021 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1081 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1141 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1201 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1261 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1321 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1381 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1441 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1501 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1561 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1621 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1681 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1741 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1801 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1861 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1921 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1981 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2041 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2101 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2161 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2221 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     2281 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2341 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2401 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2461 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2521 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2581 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2641 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2701 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2761 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     2821 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     2881 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     2941 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3001 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3061 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3121 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3181 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3241 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     3301 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3361 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3421 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3481 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3541 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3601 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3661 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     3721 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     3781 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     3841 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     3901 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     3961 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4021 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4081 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4141 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4201 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4261 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     4321 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     4381 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     4441 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     4501 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     4561 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     4621 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4681 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     4741 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     4801 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     4861 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     4921 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     4981 agatatacca tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc
     5041 ggcagccata tggctagcat gactggtgga cagcaaatgg gtcggatcat ggtgagcaag
     5101 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     5161 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     5221 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     5281 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     5341 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     5401 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     5461 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     5521 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     5581 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     5641 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     5701 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     5761 cgccactcca ccggcggcat ggacgagctg tacaagggtg cgcctggttc tgcaggctcc
     5821 gcagctggcg gatcggggat cggcaagcct tcctctcaca gtcagtatac cagcagtggt
     5881 tctgtgtctt cctcaggcag caaaagccac catagccatt cttcctcctc ttcctcatct
     5941 gcttccacct cagggaagat gaaaagcagt aaatcagaag gttcatcaag ttccaagtta
     6001 agtagcagta tgtattctag ccaggggtct tctggatcta gccagtccaa aaattcatcc
     6061 cagtctgggg ggaagccagg ctcctctccc ataaccaagc atggactgag cagtggctct
     6121 agcagcacca agatgaaacc tcaaggaaag ccatcatcac ttatgaatcc ttctttaagt
     6181 aaaccaaaca tatccccttc tcattcaagg ccacctggag gctctgacaa gcttgcctct
     6241 ccaatgaagc ctgttcctgg aactcctcca tcctctaaag ccaagtcccc tatcagttca
     6301 ggttctggtg gttctcatat gtctggaact agttcaagct ctggcatgaa gtcatcttca
     6361 gggttaggat cctcaggctc gttgtcccag aaaactcccc catcatctaa ttcctgtacg
     6421 gcatcttcct cctccttttc ctcaagtggc tcttccatgt catcctctca gaaccagcat
     6481 gggagttcta aaggaaaatc tcccagcaga aacaagaagc cgtccttgac agctgtcata
     6541 gataaactga agcatggggt tgtcaccagt ggccctgggg gtgaagaccc actggacggc
     6601 cagatggggg tgagcacaaa ttcttccagc catcctatgt cctccaaaca taacatgtca
     6661 ggaggagagt ttcagggcaa gcgtgagaaa agtgataaag acaaatcaaa ggtttccacc
     6721 tccgggagtt cagtggattc ttctaagaag acctcagagt caaaaaatgt ggggagcaca
     6781 ggtgtggcaa aaattatcat cagtaagcat gatggaggct cccctagcat taaagccaaa
     6841 gtgactttgc agaaacctgg ggaaagtagt ggagaagggc ttaggcctca aatggcttct
     6901 tctaaaaact atggctctcc actcatcagt ggttccactc caaagcatga gcgtggctct
     6961 cccagccata gtaagtcacc agcatatacc ccccagaatc tggacagtga aagtgagtca
     7021 ggctcctcca tagcagagaa atcttatcag aatagtccca gctcagacga tggtatccga
     7081 ccacttccag aatacagcac agagaaacat aagaagcaca aaaaggaaaa gaagaaagta
     7141 aaagacaaag atagggaccg agaccgggac aaagaccgag acaagaaaaa atctcatagc
     7201 atcaagccag agagttggtc caaatcaccc atctcttcag accagtcctt gtctatgaca
     7261 agtaacacaa tcttatctgc agacagaccc tcaaggctca gcccagactt tatgattggg
     7321 gaggaagatg atgatcttat ggatgtggcc ctgattggga attagtcgag caccaccacc
     7381 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     7441 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     7501 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     7561 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acac
//
