LOCUS       sequence-425826-        5875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  used to express a supercharged GFP variant that forms inclusion 
            bodies in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5875)
  AUTHORS   Yeong V, Werth EG, Brown LM, Obermeyer AC
  TITLE     Formation of Biomolecular Condensates in Bacteria by Tuning Protein 
            Electrostatics.
  JOURNAL   ACS Cent Sci. 2020 Dec 23;6(12):2301-2310. doi: 
            10.1021/acscentsci.0c01146. Epub 2020 Nov 12.
  PUBMED    33376791
REFERENCE   2  (bases 1 to 5875)
  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/acscentsci.0c01146"; journalName: "ACS Cent Sci"; date: 
            "2020-12-23- 23"; volume: "6"; issue: "12"; pages: "2301-2310"
COMMENT     Sequence Label: pET-6xHis-GFP(+36)-DL4
FEATURES             Location/Qualifiers
     source          1..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(181..273)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      354..942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     843..862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1096..1113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1128..1267
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1193..1212)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1353..1375
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1369..1560)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2132..3214)
                     /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     3176..3195
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3215..3292)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3498..3517
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3605..3624
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3605..3623
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3624..3648
                     /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             3663..3685
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3698..3715
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4445..4489
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4527..4545)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4541..4588
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4625..5080
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4712..4731)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4922..4943
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5195..5875,1..111,112..180))
                     /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     5818..5837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
       61 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
      121 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
      181 actcttcctt tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat
      241 acatatttga atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt
      301 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
      361 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      421 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      481 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
      541 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      601 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      661 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      721 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      781 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      841 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      901 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      961 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     1021 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     1081 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     1141 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     1201 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     1261 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1321 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1381 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     1441 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     1501 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     1561 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     1621 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     1681 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     1741 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     1801 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     1861 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     1921 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     1981 gcctagccgg gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg
     2041 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     2101 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2161 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2221 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     2281 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     2341 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     2401 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     2461 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     2521 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     2581 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     2641 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     2701 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     2761 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     2821 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     2881 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     2941 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     3001 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     3061 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     3121 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     3181 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     3241 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     3301 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     3361 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     3421 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     3481 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     3541 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     3601 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3661 attttgttta actttaagaa ggagatatac catgggtcat caccaccacc atcacggtgg
     3721 cgctagcaaa ggtgaacgtc tgtttcgtgg taaagtaccg atcttagtgg aattaaaggg
     3781 cgacgtgaac ggtcataaat ttagcgtgcg cggcaaaggc aaaggtgacg ctacccgtgg
     3841 taaattgacc ctgaagttta tttgcacaac aggcaaatta cccgttccgt ggcccacctt
     3901 agtgaccacc ctgacctatg gcgttcagtg cttcagtcgt taccctaaac atatgaaacg
     3961 tcacgatttt ttcaaatcag ccatgcctaa aggatatgtt caagagcgta caatcagctt
     4021 caagaaggat ggcaaatata aaacgcgtgc ggaagtgaaa tttgaaggcc gcacattagt
     4081 aaatcgtatc aaactgaaag gtcgtgactt caaagaaaaa ggcaacattt taggccataa
     4141 actgcgttat aactttaatt ctcataaggt gtatattacg gccgataaac gcaagaatgg
     4201 tatcaaggca aaattcaaaa ttcgccataa cgtgaaagac ggcagcgttc aattagcgga
     4261 tcattatcaa caaaacacgc cgattggtcg ccgcaaccac tacctgagca cccgttctaa
     4321 actgagcaaa gatccgaaag aaaaacgcga tcacatggtt ctgttagaat tcgtgaccgc
     4381 tgcaggcatt aagcacggac gcgacgaacg ctacaagtaa taatgaggta ccctcgagtc
     4441 tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc
     4501 agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     4561 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     4621 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4681 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4741 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4801 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4861 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4921 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4981 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5041 tttaacgcga attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga
     5101 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5161 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5221 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5281 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5341 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5401 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5461 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5521 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5581 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5641 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5701 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5761 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5821 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttc
//
