LOCUS       sequence-392689-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  used to express a supercharged GFP variant in E. coli.
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-GFP(+36)
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(311..1393)
                     /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     1355..1374
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1394..1471)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1677..1696
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1784..1803
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1784..1802
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1803..1827
                     /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             1842..1864
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1877..1894
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2642..2686
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2724..2742)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2738..2785
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2822..3277
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2909..2928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3119..3140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3392..4183,4184..4252))
                     /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     4015..4034
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4253..4345)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4426..5014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4915..4934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5168..5185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5200..5339
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5265..5284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5425..5447
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5441..5632)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
       61 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
      121 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
      181 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
      241 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
      301 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
      361 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
      421 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
      481 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
      541 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
      601 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
      661 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
      721 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
      781 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
      841 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
      901 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
      961 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     1021 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     1081 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     1141 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     1201 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     1261 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     1321 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     1381 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     1441 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     1501 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     1561 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     1621 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     1681 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     1741 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata
     1801 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1861 gagatatacc atgggtcatc accaccacca tcacggtggc gctagcaaag gtgaacgtct
     1921 gtttcgtggt aaagtaccga tcttagtgga attaaagggc gacgtgaacg gtcataaatt
     1981 tagcgtgcgc ggcaaaggca aaggtgacgc tacccgtggt aaattgaccc tgaagtttat
     2041 ttgcacaaca ggcaaattac ccgttccgtg gcccacctta gtgaccaccc tgacctatgg
     2101 cgttcagtgc ttcagtcgtt accctaaaca tatgaaacgt cacgattttt tcaaatcagc
     2161 catgcctaaa ggatatgttc aagagcgtac aatcagcttc aagaaggatg gcaaatataa
     2221 aacgcgtgcg gaagtgaaat ttgaaggccg cacattagta aatcgtatca aactgaaagg
     2281 tcgtgacttc aaagaaaaag gcaacatttt aggccataaa ctgcgttata actttaattc
     2341 tcataaggtg tatattacgg ccgataaacg caagaatggt atcaaggcaa aattcaaaat
     2401 tcgccataac gtgaaagacg gcagcgttca attagcggat cattatcaac aaaacacgcc
     2461 gattggtcgc gggcctgtac tgttacctcg caaccactac ctgagcaccc gttctaaact
     2521 gagcaaagat ccgaaagaaa aacgcgatca catggttctg ttagaattcg tgaccgctgc
     2581 aggcattaag cacggacgcg acgaacgcta caagtaataa tgaggtaccc tcgagtctgg
     2641 taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc
     2701 ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     2761 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     2821 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2881 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2941 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3001 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3061 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3121 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3181 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3241 aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat
     3301 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3361 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3421 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3481 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3541 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3601 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3661 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3721 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3781 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3841 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3901 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3961 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4021 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4081 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4141 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4201 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4261 tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4321 gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc
     4381 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4441 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4501 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4561 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4621 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4681 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4741 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4801 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4861 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4921 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4981 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5041 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5101 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5161 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     5221 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     5281 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     5341 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5401 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5461 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     5521 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     5581 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     5641 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5701 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5761 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     5821 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     5881 ggaacataat ggt
//
