LOCUS       sequence-405664-        6716 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 6716)
  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 6716)
  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)-L1
FEATURES             Location/Qualifiers
     source          1..6716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1023..1214
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1208..1230)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1316..1458
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1371..1390
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1473..1490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1644..2232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1724..1743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2313..2405
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2406..2474,2475..3266)
                     /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(2624..2643)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3381..3836)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3518..3539)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3730..3749
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3873..3920)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3916..3934
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3972..4016)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(4062..4766)
                     /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(4337..4356)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4606..4624
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(4773..4790)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5584..5601)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5614..5636
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5651..5675
                     /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(5675..5694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5676..5694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5782..5801)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6007..6084
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6085..6716,1..451)
                     /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(6104..6123)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
       61 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
      121 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt
      181 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
      241 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
      301 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
      361 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
      421 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
      481 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
      541 tccggtgggc gcggggcatg actagcatga tcgtgctcct gtcgttgagg acccggctag
      601 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
      661 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
      721 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
      781 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
      841 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
      901 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
      961 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     1021 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     1081 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     1141 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     1201 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     1261 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     1321 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     1381 gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg
     1441 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1501 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1561 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1621 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1681 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1741 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1801 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1861 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1921 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1981 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2041 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     2101 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2161 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2221 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2281 aactcacgtt aagggatttt ggtcatgacc tatttgttta tttttctaaa tacattcaaa
     2341 tatgtatccg ctcatgagac aataaccctg ataaatgctt caatcatgat tgaaaaagga
     2401 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2461 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2521 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2581 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2641 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2701 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2761 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2821 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2881 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2941 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3001 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3061 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3121 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3181 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3241 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3301 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3361 tcatgccatc gtgccgccag aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa
     3421 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3481 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     3541 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     3601 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     3661 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     3721 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     3781 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     3841 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     3901 gccccaaggg gttatgctag ttattgctca gcggtggcag cagcctaggt taattaagct
     3961 gcgctagtag acgagtccat gtgctggcgt tcaaatttcg cagcagcggt ttctttacca
     4021 gactcgagtc attactttcg agatttcttg cctcttcgtc ccttgtacag ctcgtccatg
     4081 ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg
     4141 ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg cgccgggcag ctgcacgggc
     4201 ttcttggcct tgtaggtggt cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc
     4261 agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag
     4321 gcctcccagc ccatggtctt cttctgcatt acggggccgt cggaggggaa gttggtgccg
     4381 cgcagcttca ccttgtagat gaactcgccg tcctggaggg aggagtcctg ggtcacggtc
     4441 accacgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac
     4501 agcttcaagt agtcggggat gtcggcgggg tgcttcacgt aggccttgga gccgtacatg
     4561 aactgagggg acaggatgtc ccaggcgaag ggcagggggc cacccttggt caccttcagc
     4621 ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg
     4681 tggccgttca cggagccctc catgtgcacc ttgaagcgca tgaactcctt gatgatggcc
     4741 atgttatcct cctcgccctt gctcacgcca ccgtgatggt ggtggtgatg acccatatgt
     4801 atatctcctt cttaaagtta attatttctt ccttctcctc tttttcttcc ttctcctctt
     4861 tttcttgtac agctcgtcca ttccgtgagt aatgcctgca gcggtcacga attctaacag
     4921 aaccatgtga tcgcgttttt cgttcggatc tttgctcagt gctgactggg tgctcaggta
     4981 gtggttgtca ggtaacagta caggcccgtc accaatcggc gtgttttgtt gataatgatc
     5041 cgctaattga acgctgccgt cttccacgtt atggcgaatt ttgaaatttg ccttgatacc
     5101 gtttttctgt ttatcggccg taatatacac gttatgagaa ttaaagttat attccagctt
     5161 atggcctaaa atgttgccgt cttctttgaa gtcgatacct ttcagttcga tacggtttac
     5221 taatgtgtcg ccttcaaatt tcacttccgc acgcgtttta taggtgccat cgtccttgaa
     5281 gctgattgta cgctcttgaa catatccttc aggcatggct gatttgaaaa aatcgtgttg
     5341 tttcatatga tctgggtaac gactgaagca ctgaacgcca taggtcaggg tggtcactaa
     5401 ggtgggccac ggaacgggta atttgcctgt tgtgcaaata aacttcaggg tcaatttacc
     5461 attggtagcg tcaccttcgc cttcgccgcg cacgctaaat ttgtgaccgt tcacgtcgcc
     5521 gtctaattcc actaagatcg gtactacacc cgtaaacagc tcttcacctt tgctagcgcc
     5581 accgtgatgg tggtggtgat gacccatggt atatctcctt cttaaagtta aacaaaatta
     5641 tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg
     5701 cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     5761 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     5821 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     5881 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
     5941 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     6001 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     6061 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     6121 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     6181 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     6241 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     6301 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     6361 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     6421 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     6481 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     6541 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     6601 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     6661 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattca
//
