LOCUS       sequence-405665-        6743 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 6743)
  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 6743)
  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)-L2
FEATURES             Location/Qualifiers
     source          1..6743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1266..1457
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1451..1473)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1559..1701
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1614..1633
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1716..1733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1887..2475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1967..1986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2556..2648
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2649..2717,2718..3509)
                     /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(2867..2886)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3624..4079)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3761..3782)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3973..3992
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4116..4163)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4159..4177
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4215..4259)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(4332..5036)
                     /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(4607..4626)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4876..4894
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(5043..5060)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5854..5871)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5884..5906
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5921..5945
                     /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(5945..5964)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5946..5964)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6052..6071)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6277..6354
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6355..6743,1..694)
                     /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(6374..6393)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
       61 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
      121 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
      181 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
      241 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
      301 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
      361 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg acatctcggt
      421 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
      481 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
      541 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
      601 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
      661 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
      721 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
      781 ccttccggtg ggcgcggggc atgactagca tgatcgtgct cctgtcgttg aggacccggc
      841 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
      901 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
      961 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     1021 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     1081 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     1141 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     1201 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     1261 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     1321 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     1381 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     1441 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     1501 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     1561 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     1621 agagcagatt gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta
     1681 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2521 gaaaactcac gttaagggat tttggtcatg acctatttgt ttatttttct aaatacattc
     2581 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaatcat gattgaaaaa
     2641 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2701 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2761 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2821 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2881 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2941 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3001 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3061 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3121 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3181 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3241 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3301 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3361 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3421 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3481 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3541 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3601 atctcatgcc atcgtgccgc cagaaattgt aaacgttaat attttgttaa aattcgcgtt
     3661 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3721 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3781 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3841 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3901 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     3961 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4021 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4081 ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
     4141 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccta ggttaattaa
     4201 gctgcgctag tagacgagtc catgtgctgg cgttcaaatt tcgcagcagc ggtttcttta
     4261 ccagactcga gtcattactt tcgagatttc ttgcctcttc gtccctttcg agatttcttg
     4321 cctcttcgtc ccttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt
     4381 tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg
     4441 acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctca
     4501 gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg
     4561 ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt
     4621 acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg
     4681 tcctggaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg
     4741 cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg
     4801 tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag
     4861 ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg
     4921 ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
     4981 ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcacgcca
     5041 ccgtgatggt ggtggtgatg acccatatgt atatctcctt cttaaagtta attatttctt
     5101 ccttctcctc tttttcttcc ttctcctctt tttcttgtac agctcgtcca ttccgtgagt
     5161 aatgcctgca gcggtcacga attctaacag aaccatgtga tcgcgttttt cgttcggatc
     5221 tttgctcagt gctgactggg tgctcaggta gtggttgtca ggtaacagta caggcccgtc
     5281 accaatcggc gtgttttgtt gataatgatc cgctaattga acgctgccgt cttccacgtt
     5341 atggcgaatt ttgaaatttg ccttgatacc gtttttctgt ttatcggccg taatatacac
     5401 gttatgagaa ttaaagttat attccagctt atggcctaaa atgttgccgt cttctttgaa
     5461 gtcgatacct ttcagttcga tacggtttac taatgtgtcg ccttcaaatt tcacttccgc
     5521 acgcgtttta taggtgccat cgtccttgaa gctgattgta cgctcttgaa catatccttc
     5581 aggcatggct gatttgaaaa aatcgtgttg tttcatatga tctgggtaac gactgaagca
     5641 ctgaacgcca taggtcaggg tggtcactaa ggtgggccac ggaacgggta atttgcctgt
     5701 tgtgcaaata aacttcaggg tcaatttacc attggtagcg tcaccttcgc cttcgccgcg
     5761 cacgctaaat ttgtgaccgt tcacgtcgcc gtctaattcc actaagatcg gtactacacc
     5821 cgtaaacagc tcttcacctt tgctagcgcc accgtgatgg tggtggtgat gacccatggt
     5881 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     5941 attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg atcctctacg
     6001 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     6061 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     6121 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     6181 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     6241 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     6301 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     6361 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     6421 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     6481 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     6541 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     6601 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     6661 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     6721 atcattaact atccgctgga tga
//
