LOCUS       sequence-405663-        6689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expressed fluorescent proteins with charge-patterned cationic 
            peptide to promote multi-protein biomolecular condensate formation 
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6689)
  AUTHORS   Liao J, Yeong V, Obermeyer AC
  TITLE     Charge-Patterned Disordered Peptides Tune Intracellular Phase 
            Separation in Bacteria.
  JOURNAL   ACS Synth Biol. 2024 Feb 3. doi: 10.1021/acssynbio.3c00564.
  PUBMED    38308651
REFERENCE   2  (bases 1 to 6689)
  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: "ACS Synth 
            Biol. 2024 Feb 3. doi: 10.1021/acssynbio.3c00564."
COMMENT     Sequence Label: pET-6xHis-GFP(-6)-H2_mCherry(-6)
FEATURES             Location/Qualifiers
     source          1..6689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             889..1080
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1074..1096)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1182..1324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1237..1256
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1339..1356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1510..2098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1590..1609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2179..2271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2272..2340,2341..3132)
                     /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(2490..2509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3247..3702)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3384..3405)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3596..3615
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3739..3786)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3782..3800
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3838..3882)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(3898..4605)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4176..4195)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4445..4463
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(4612..4629)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5423..5440)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5453..5475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5490..5514
                     /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)."
     primer_bind     complement(5514..5533)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5515..5533)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5621..5640)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5846..5923
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5924..6689,1..317)
                     /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     complement(5943..5962)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata
       61 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
      121 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
      181 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
      241 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      301 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
      361 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
      421 ggcatgacta gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
      481 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
      541 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
      601 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
      661 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
      721 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
      781 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
      841 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
      901 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
      961 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     1021 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     1081 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     1141 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     1201 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     1261 gagtgcacca tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     1321 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1381 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1441 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1681 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1921 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     1981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2161 gattttggtc atgacctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2221 tgagacaata accctgataa atgcttcaat catgattgaa aaaggaagag tatgagtatt
     2281 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2341 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2401 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2461 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2521 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2581 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2641 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2701 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2761 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2821 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2881 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2941 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3001 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3061 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3121 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3181 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gccatcgtgc
     3241 cgccagaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3301 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     3361 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3421 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3481 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3541 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3601 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3661 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga
     3721 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
     3781 tgctagttat tgctcagcgg tggcagcagc ctaggttaat taagctgcgc tagtagacga
     3841 gtccatgtgc tggcgttcaa atttcgcagc agcggtttct ttaccagact cgagtcatta
     3901 cttgtacagc tcgtccatgc cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc
     3961 cacgatggtg tagtcctcgt tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc
     4021 gccgggcagc tgcacgggct tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg
     4081 gccgccgtcc ttcagcttca gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg
     4141 gtacatccgc tcggaggagg cctcccagcc catggtcttc ttctgcatta cggggccgtc
     4201 ggaggggaag ttggtgccgc gcagcttcac cttgtagatg aactcgccgt cctggaggga
     4261 ggagtcctgg gtcacggtca ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt
     4321 gaagccctcg gggaaggaca gcttcaagta gtcggggatg tcggcggggt gcttcacgta
     4381 ggccttggag ccgtacatga actgagggga caggatgtcc caggcgaagg gcagggggcc
     4441 acccttggtc accttcagct tggcggtctg ggtgccctcg taggggcggc cctcgccctc
     4501 gccctcgatc tcgaactcgt ggccgttcac ggagccctcc atgtgcacct tgaagcgcat
     4561 gaactccttg atgatggcca tgttatcctc ctcgcccttg ctcacgccac cgtgatggtg
     4621 gtggtgatga cccatatgta tatctccttc ttaaagttaa ttatttcttc cttctcctct
     4681 ttttcttcct tctcctcttt ttcttgtaca gctcgtccat tccgtgagta atgcctgcag
     4741 cggtcacgaa ttctaacaga accatgtgat cgcgtttttc gttcggatct ttgctcagtg
     4801 ctgactgggt gctcaggtag tggttgtcag gtaacagtac aggcccgtca ccaatcggcg
     4861 tgttttgttg ataatgatcc gctaattgaa cgctgccgtc ttccacgtta tggcgaattt
     4921 tgaaatttgc cttgataccg tttttctgtt tatcggccgt aatatacacg ttatgagaat
     4981 taaagttata ttccagctta tggcctaaaa tgttgccgtc ttctttgaag tcgatacctt
     5041 tcagttcgat acggtttact aatgtgtcgc cttcaaattt cacttccgca cgcgttttat
     5101 aggtgccatc gtccttgaag ctgattgtac gctcttgaac atatccttca ggcatggctg
     5161 atttgaaaaa atcgtgttgt ttcatatgat ctgggtaacg actgaagcac tgaacgccat
     5221 aggtcagggt ggtcactaag gtgggccacg gaacgggtaa tttgcctgtt gtgcaaataa
     5281 acttcagggt caatttacca ttggtagcgt caccttcgcc ttcgccgcgc acgctaaatt
     5341 tgtgaccgtt cacgtcgccg tctaattcca ctaagatcgg tactacaccc gtaaacagct
     5401 cttcaccttt gctagcgcca ccgtgatggt ggtggtgatg acccatggta tatctccttc
     5461 ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata
     5521 gtgagtcgta ttaatttcgc gggatcgaga tcgatctcga tcctctacgc cggacgcatc
     5581 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     5641 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     5701 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     5761 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     5821 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     5881 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     5941 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     6001 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     6061 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     6121 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     6181 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     6241 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     6301 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     6361 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     6421 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     6481 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     6541 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     6601 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     6661 cgatcagatg gcgctgggcg caatgcgcg
//
