LOCUS       sequence-449510-        5970 bp    DNA     circular SYN 14-APR-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 5970)
  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 5970)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-LSSmCherry1(-13)
FEATURES             Location/Qualifiers
     source          1..5970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             178..885
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMTIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVELRGTNFPSDGPVMQCRTMGLEASTERMYPEDGALKG
                     ESKERLKLKDGGHYDAEVKTTYKAKEPVQLPGAYNVDIKLDILSHNEDYTIVEQYERSE
                     GRHSTGGMDELYK"
     primer_bind     complement(320..338)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             895..912
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(965..983)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      979..1026
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1063..1518
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1150..1169)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1360..1381
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1610..2425)
                     /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(1610..2425)
                     /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     2333..2352
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2417..2436
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2547..3135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3036..3055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3289..3306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3321..3460
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3386..3405)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3546..3568
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3562..3753)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4562..5644)
                     /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     5606..5625
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5645..5722)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5928..5947
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atataccatg ggccatcatc atcatcatca tggcggcgtg
      181 agcaagggcg aggaggataa catgaccatc atcaaggagt tcatgcgctt caaggtgcac
      241 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
      301 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
      361 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
      421 gacatccccg actacttgaa gctgtccttc cccgagggct tcaattggga gcgcgtgatg
      481 aactttgagg acggcggtgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
      541 ttcatctaca aggtggagct gcgcggcacc aacttcccct ctgacggccc cgtaatgcag
      601 tgtcgtacca tgggcttgga ggccagtact gagcggatgt accccgagga cggcgccctg
      661 aagggcgaga gtaaggagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
      721 aagaccacct acaaggccaa ggagcccgtg cagctgcccg gcgcctacaa cgtcgacatc
      781 aagttggaca tcctttccca caacgaggac tacaccatcg tggaacagta cgaacgctcc
      841 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaataact cgagcaccac
      901 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      961 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1021 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1081 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1141 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1201 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1261 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1321 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1381 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1441 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1501 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1561 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1621 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1681 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1741 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1801 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1861 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1921 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1981 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2041 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2101 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2161 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2221 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     2281 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     2341 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2401 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2461 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3121 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3181 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3241 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3301 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3361 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3421 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3481 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3541 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3601 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3661 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3721 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3781 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3841 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     3901 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     3961 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4021 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4081 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4141 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4201 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4261 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4321 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4381 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4441 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4501 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4561 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4621 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4681 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4741 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4801 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     4861 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4921 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4981 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5041 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5101 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5161 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5221 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5281 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5341 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5401 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5461 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5521 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5581 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5641 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5701 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5761 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     5821 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     5881 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     5941 atataggcgc cagcaaccgc acctgtggcg
//
