LOCUS       sequence-449508-        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent protein to reconstruct the mixture that mimics the 
            charge profile of E. coli lysate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  AUTHORS   Ahn SY, Obermeyer AC
  TITLE     Selectivity of Complex Coacervation in Multiprotein Mixtures.
  JOURNAL   JACS Au. 2024 Sep 17;4(10):3800-3812. doi: 10.1021/jacsau.4c00399. 
            eCollection 2024 Oct 28.
  PUBMED    39483238
REFERENCE   2  (bases 1 to 6954)
  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"; doi: 
            "10.1021/jacsau.4c00399"; journalName: "JACS Au"; date: "2024-09-17-
            17"; volume: "4"; issue: "10"; pages: "3800-3812"
COMMENT     Sequence Label: pET28-a(+)-6xHis-miRFP(-5)-HO1
FEATURES             Location/Qualifiers
     source          1..6954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..90
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(110..187)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     393..412
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     496..515
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        496..514
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    515..539
                     /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             554..576
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             589..606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             613..1560
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 682 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP682
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="AEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSD
                     NLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIG
                     SWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITG
                     FDRVMIYRFASDFSGVVIAEDRCAEVESKLGLHYPASAVPAQARRLYTINPVRIIPDIN
                     YRPVPVTPDLNPVTGRPIDLSFAILRSVSPCHLEFMRNIGMHGTMSISILRGERLWGLI
                     VCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE"
     CDS             2314..2331
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2384..2402)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2398..2445
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2482..2937
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2569..2588)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2779..2800
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3029..3844)
                     /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(3029..3844)
                     /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     3752..3771
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3836..3855
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3966..4554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4455..4474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4708..4725
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4740..4879
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4805..4824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4965..4987
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4981..5172)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5981..6954,1..109))
                     /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"
ORIGIN
        1 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
       61 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
      121 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
      181 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
      241 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
      301 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
      361 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
      421 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
      481 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
      541 ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcca tcatcatcat
      601 catcatggcg gcgcggaagg atccgtcgcc aggcagcctg acctcttgac ctgcgacgat
      661 gagccgatcc atatccccgg tgccatccaa ccgcatggac tgctgctcgc cctcgccgcc
      721 gacatgacga tcgttgccgg cagcgacaac cttcccgaac tcaccggact ggcgatcggc
      781 gccctgatcg gccgctctgc ggccgatgtc ttcgactcgg agacgcacaa ccgtctgacg
      841 atcgccttgg ccgagcccgg ggcggccgtc ggagcaccga tcactgtcgg cttcacgatg
      901 cgaaaggacg caggcttcat cggctcctgg catcgccatg atcagctcat cttcctcgag
      961 ctcgagcctc cccagcggga cgtcgccgag ccgcaggcgt tcttccgccg caccaacagc
     1021 gccatccgcc gcctgcaggc cgccgaaacc ttggaaagcg cctgcgccgc cgcggcgcaa
     1081 gaggtgcgga agattaccgg cttcgatcgg gtgatgatct atcgcttcgc ctccgacttc
     1141 agcggggtgg tgatcgcaga ggatcgatgc gccgaggtcg agtcaaaact aggcctgcac
     1201 tatcctgcct cagcggtgcc ggcgcaggcc cgtcggctct ataccatcaa cccggtacgg
     1261 atcattcccg atatcaatta tcggccggtg ccggtcaccc cagacctcaa tccggtcacc
     1321 gggcggccga ttgatcttag cttcgccatc ctgcgcagcg tctcgccctg ccatttggag
     1381 ttcatgcgca acataggcat gcacggcacg atgtcgatct cgattttgcg cggcgagcga
     1441 ctgtggggat tgatcgtttg ccatcaccga acgccgtact acgtcgatct cgatggccgc
     1501 caagcctgca agcgggtcgc cgagcggctg gccacgcaga tcggcgtgat ggaagagtga
     1561 aagaaggaga tatacatacc catgagtgtc aacttagctt cccagttgcg ggaagggacg
     1621 aaaaaatccc actccatggc ggagaacgtc ggctttgtca aatgcttcct caagggcgtt
     1681 gtcgagaaaa attcctaccg taagctggtt ggcaatctct actttgtcta cagtgccatg
     1741 gaagaggaaa tggcaaaatt taaggaccat cccatcctca gccacattta cttccccgaa
     1801 ctcaaccgca aacaaagcct agagcaagac ctgcaattct attacggctc caactggcgg
     1861 caagaagtga aaatttctgc cgctggccaa gcctatgtgg accgagtccg gcaagtggcc
     1921 gctacggccc ctgaattgtt ggtggcccat tcctacaccc gttacctggg ggatctttcc
     1981 ggcggtcaaa ttctcaagaa aattgcccaa aatgccatga atctccacga tggtggcaca
     2041 gctttctatg aatttgccga cattgatgac gaaaaggctt ttaaaaatac ctaccgtcaa
     2101 gctatgaatg atctgcccat tgaccaagcc accgccgaac ggattgtgga tgaagccaat
     2161 gacgcctttg ccatgaacat gaaaatgttc aacgaacttg aaggcaacct gatcaaggcg
     2221 atcggcatta tggtgttcaa cagcctcacc cgtcgccgca gtcaaggcag caccgaagtt
     2281 ggcctcgcca cctccgaagg ctagtaactc gagcaccacc accaccacca ctgagatccg
     2341 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2401 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2461 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2521 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2581 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2641 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2701 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2761 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2821 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2881 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2941 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3001 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     3061 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3121 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3181 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3241 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     3301 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3361 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3421 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3481 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3541 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3601 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3661 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3721 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3781 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3841 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     3901 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3961 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4021 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4081 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4141 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4201 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4261 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4321 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4381 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4441 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4501 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4561 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4621 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4681 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4741 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4801 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4861 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4921 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4981 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5041 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5101 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5161 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5221 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5281 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5341 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5401 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5461 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5521 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5581 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     5641 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     5701 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5761 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5821 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5881 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5941 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6001 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6061 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     6121 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     6181 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     6241 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     6301 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     6361 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     6421 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     6481 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     6541 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     6601 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     6661 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     6721 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     6781 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6841 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6901 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actt
//
