LOCUS       sequence-405667-        6770 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 6770)
  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 6770)
  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)-L3
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    92..234
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     147..166
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(249..266)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(420..1008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(500..519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1089..1181
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1182..1250,1251..2042)
                     /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(1400..1419)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2157..2612)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2294..2315)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2506..2525
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2649..2696)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2692..2710
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2748..2792)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(2892..3596)
                     /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(3167..3186)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3436..3454
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3603..3620)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4414..4431)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4444..4466
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4481..4505
                     /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(4505..4524)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4506..4524)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4612..4631)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4837..4914
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4915..5997
                     /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(4934..4953)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6569..6760
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(join(6754..6770,1..6))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
       61 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
      121 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
      181 tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
      241 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      301 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      361 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      421 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      481 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      541 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      601 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      661 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      721 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      781 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      841 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
      901 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
      961 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1021 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1081 atgacctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1141 accctgataa atgcttcaat catgattgaa aaaggaagag tatgagtatt caacatttcc
     1201 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1261 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1321 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1381 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1441 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1501 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1561 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1621 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1681 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     1741 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1801 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     1861 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     1921 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     1981 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2041 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2101 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gccatcgtgc cgccagaaat
     2161 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2221 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2281 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2341 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2401 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2461 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2521 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2581 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct
     2641 cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat
     2701 tgctcagcgg tggcagcagc ctaggttaat taagctgcgc tagtagacga gtccatgtgc
     2761 tggcgttcaa atttcgcagc agcggtttct ttaccagact cgagttatta cttccgcgat
     2821 ttcttccctc ttcgaccctt ccgcgatttc ttccctcttc gaccctttcg agatttcttg
     2881 cctcttcgtc ccttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt
     2941 tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg
     3001 acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctca
     3061 gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg
     3121 ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt
     3181 acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg
     3241 tcctggaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg
     3301 cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg
     3361 tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag
     3421 ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg
     3481 ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
     3541 ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcacgcca
     3601 ccgtgatggt ggtggtgatg acccatatgt atatctcctt cttaaagtta attatttctt
     3661 ccttctcctc tttttcttcc ttctcctctt tttcttgtac agctcgtcca ttccgtgagt
     3721 aatgcctgca gcggtcacga attctaacag aaccatgtga tcgcgttttt cgttcggatc
     3781 tttgctcagt gctgactggg tgctcaggta gtggttgtca ggtaacagta caggcccgtc
     3841 accaatcggc gtgttttgtt gataatgatc cgctaattga acgctgccgt cttccacgtt
     3901 atggcgaatt ttgaaatttg ccttgatacc gtttttctgt ttatcggccg taatatacac
     3961 gttatgagaa ttaaagttat attccagctt atggcctaaa atgttgccgt cttctttgaa
     4021 gtcgatacct ttcagttcga tacggtttac taatgtgtcg ccttcaaatt tcacttccgc
     4081 acgcgtttta taggtgccat cgtccttgaa gctgattgta cgctcttgaa catatccttc
     4141 aggcatggct gatttgaaaa aatcgtgttg tttcatatga tctgggtaac gactgaagca
     4201 ctgaacgcca taggtcaggg tggtcactaa ggtgggccac ggaacgggta atttgcctgt
     4261 tgtgcaaata aacttcaggg tcaatttacc attggtagcg tcaccttcgc cttcgccgcg
     4321 cacgctaaat ttgtgaccgt tcacgtcgcc gtctaattcc actaagatcg gtactacacc
     4381 cgtaaacagc tcttcacctt tgctagcgcc accgtgatgg tggtggtgat gacccatggt
     4441 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     4501 attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg
     4561 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     4621 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     4681 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     4741 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     4801 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     4861 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     4921 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     4981 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     5041 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     5101 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     5161 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     5221 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     5281 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     5341 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     5401 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     5461 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     5521 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     5581 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     5641 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     5701 cgcgttggtg cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     5761 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     5821 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     5881 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5941 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     6001 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     6061 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta gcatgatcgt
     6121 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     6181 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     6241 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     6301 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     6361 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     6421 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     6481 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     6541 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     6601 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     6661 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     6721 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
//
