LOCUS       sequence-392681-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  used to express a superfolder GFP in E. coli for comparison to 
            supercharged variants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5893)
  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 5893)
  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-sfGFP
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     756..775
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(795..872)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1078..1097
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1185..1204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1185..1203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1204..1228
                     /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             1243..1265
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1278..1295
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2043..2087
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2125..2143)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2139..2186
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2223..2678
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2310..2329)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2520..2541
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2793..3584,3585..3653))
                     /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     3416..3435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3654..3746)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3827..4415
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4316..4335
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4569..4586
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4601..4740
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4666..4685)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4826..4848
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4842..5033)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5605..5893,1..794))
                     /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 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
       61 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
      121 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
      181 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
      241 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
      301 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
      361 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
      421 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
      481 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
      541 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
      601 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
      661 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
      721 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
      781 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
      841 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
      901 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
      961 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     1021 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     1081 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     1141 gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat
     1201 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1261 ggagatatac catgggtcat caccaccacc atcacggtgg cgctagcaaa ggtgaagagc
     1321 tgtttacggg tgtagtaccg atcttagtgg aattagacgg cgacgtgaac ggtcacaaat
     1381 ttagcgtgcg cggcgaaggc gaaggtgacg ctaccaatgg taaattgacc ctgaagttta
     1441 tttgcacaac aggcaaatta cccgttccgt ggcccacctt agtgaccacc ctgacctatg
     1501 gcgttcagtg cttcagtcgt tacccagatc atatgaaaca acacgatttt ttcaaatcag
     1561 ccatgcctga aggatatgtt caagagcgta caatcagctt caaggacgat ggcacctata
     1621 aaacgcgtgc ggaagtgaaa tttgaaggcg acacattagt aaaccgtatc gaactgaaag
     1681 gtatcgactt caaagaagac ggcaacattt taggccataa gctggaatat aactttaatt
     1741 ctcataacgt gtatattacg gccgataaac agaaaaacgg tatcaaggca aatttcaaaa
     1801 ttcgccataa cgtggaagac ggcagcgttc aattagcgga tcattatcaa caaaacacgc
     1861 cgattggtga cgggcctgta ctgttacctg acaaccacta cctgagcacc cagtcagcac
     1921 tgagcaaaga tccgaacgaa aaacgcgatc acatggttct gttagaattc gtgaccgctg
     1981 caggcattac tcacggaatg gacgaactct acaagtaata atgaggtacc ctcgagtctg
     2041 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     2101 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2161 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2221 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2281 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2341 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2401 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2461 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2521 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2581 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2641 taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga tggcatgaga
     2701 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2761 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2821 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2881 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2941 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3001 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3061 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3121 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3181 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3241 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3301 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3361 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3421 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3481 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3541 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3601 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3661 ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3721 tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt
     3781 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3841 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3901 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3961 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4021 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4081 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4141 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4201 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4261 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4321 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4381 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4441 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4501 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4561 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4621 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4681 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4741 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4801 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4861 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     4921 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4981 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     5041 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     5101 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     5161 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     5221 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     5281 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     5341 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5401 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5461 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
     5521 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5581 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5641 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5701 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5761 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5821 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5881 cgagatgtcc gca
//
