LOCUS       sequence-392686-        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(-30)
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     522..541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(760..852)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      933..1521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1422..1441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1675..1692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1707..1846
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1772..1791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1932..1954
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1948..2139)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2711..3793)
                     /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     3755..3774
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3794..3871)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4077..4096
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4184..4203
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4184..4202
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4203..4227
                     /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             4242..4264
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4277..4294
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5042..5086
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5124..5142)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5138..5185
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5222..5677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5309..5328)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5519..5540
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5792..5893,1..690,691..759))
                     /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"
ORIGIN
        1 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
       61 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
      121 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      181 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
      241 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
      301 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
      361 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
      421 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
      481 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
      541 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
      601 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
      661 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
      721 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
      781 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
      841 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
      901 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
      961 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1021 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     1081 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1141 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1201 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     1261 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     1321 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     1381 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     1441 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     1501 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     1561 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     1621 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     1681 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     1741 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     1801 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     1861 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     1921 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     1981 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     2041 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     2101 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     2161 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     2221 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     2281 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     2341 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     2401 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     2461 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     2521 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     2581 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     2641 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     2701 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2761 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     2821 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     2881 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     2941 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     3001 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     3061 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     3121 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     3181 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     3241 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     3301 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     3361 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     3421 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     3481 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     3541 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     3601 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     3661 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     3721 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     3781 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     3841 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     3901 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     3961 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     4021 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     4081 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     4141 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata
     4201 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     4261 gagatatacc atgggtcatc accaccacca tcacggtggc gctagcaaag gtgaagagct
     4321 gtttgacggt gtagtaccga tcttagtgga attagacggc gacgtgaacg gtcacgaatt
     4381 tagcgtgcgc ggcgagggcg aaggtgacgc taccgagggt gaattgaccc tgaagtttat
     4441 ttgcacaaca ggcgaattac ccgttccgtg gcccacctta gtgaccaccc tgacctatgg
     4501 cgttcagtgc ttcagtgatt acccagatca tatggatcaa cacgattttt tcaaatcagc
     4561 catgcctgaa ggatatgttc aagagcgtac aatcagcttc aaggacgatg gcacctataa
     4621 aacgcgtgcg gaagtgaaat ttgaaggcga cacattagta aaccgtatcg aactgaaagg
     4681 tatcgacttc aaagaagacg gcaacatttt aggccataag ctggaatata actttaattc
     4741 tcatgacgtg tatattacgg ccgataaaca ggaaaacggt atcaaggcag aatttgaaat
     4801 tcgccataac gtggaggacg gcagcgttca attagcggat cattatcaac aaaacacgcc
     4861 gattggtgat gggcctgtac tgttacctga cgatcactac ctgagcacgg agtcagccct
     4921 gagcaaagat ccgaacgaag accgcgatca catggttctg ttagaattcg tgaccgctgc
     4981 aggcattgat catggaatgg acgagctgta caagtaataa tgaggtaccc tcgagtctgg
     5041 taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc
     5101 ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     5161 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     5221 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5281 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5341 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5401 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5461 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5521 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5581 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5641 aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat
     5701 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5761 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5821 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5881 ctacgatacg gga
//
