LOCUS       sequence-392679-        5893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  used to express a neutral 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(0)
FEATURES             Location/Qualifiers
     source          1..5893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     158..180
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(174..365)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(937..2019)
                     /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     1981..2000
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2020..2097)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2303..2322
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2410..2429
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2410..2428
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2429..2453
                     /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             2468..2490
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2503..2520
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3268..3312
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3350..3368)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3364..3411
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3448..3903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3535..3554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3745..3766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4018..4809,4810..4878))
                     /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     4641..4660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4879..4971)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5052..5640
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5541..5560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5794..5811
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(5826..5893,1..72)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(join(5891..5893,1..17))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
       61 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      121 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      181 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
      241 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
      301 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
      361 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
      421 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
      481 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
      541 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
      601 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
      661 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
      721 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
      781 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
      841 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
      901 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      961 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1021 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     1081 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     1141 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     1201 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     1261 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     1321 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     1381 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     1441 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     1501 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     1561 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     1621 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     1681 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     1741 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     1801 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     1861 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     1921 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     1981 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     2041 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     2101 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     2161 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     2221 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     2281 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     2341 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
     2401 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     2461 aaataatttt gtttaacttt aagaaggaga tataccatgg gtcatcacca ccaccatcac
     2521 ggtggcgcta gcaaaggtga acgtctgttt actggtgtag taccgatctt agtggaatta
     2581 gacggcgacg tgaacggtca taaatttagc gtgcgcggcg agggcgaagg tgacgctacc
     2641 aatggtaaat tgaccctgaa gtttatttgc acaacaggca aattacccgt tccgtggccc
     2701 accttagtga ccaccctgac ctatggcgtt cagtgcttca gtcgttaccc tgatcatatg
     2761 aaacaacacg attttttcaa atcagccatg cctgaaggat atgttcaaga gcgtacaatc
     2821 agcttcaagg acgatggcac ctataaaacg cgtgcggaag tgaaatttga aggcgacaca
     2881 ttagtaaatc gtatcgaact gaaaggtcgt gacttcaaag aagacggcaa cattttaggc
     2941 cataaactgg aatataactt taattctcat aacgtgtata ttacggccga taaacagaag
     3001 aatggtatca aggcaaattt caaaattcgc cataacgtgg aggacggcag cgttcaatta
     3061 gcggatcatt atcaacaaaa cacgccgatt ggtgatgggc ctgtactgtt acctcgcaac
     3121 cactacctga gcacccaatc tgccctgagc aaagatccga aagaaaaacg cgatcacatg
     3181 gttctgttag aattcgtgac cgctgcaggc attacgcacg gaatggacga acgctacaag
     3241 taataatgag gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag
     3301 cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa
     3361 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     3421 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     3481 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3541 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3601 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3661 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3721 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3781 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3841 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3901 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3961 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4021 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4081 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4141 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4201 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4261 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4321 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4381 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4441 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4501 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4561 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4621 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4681 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4741 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4801 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4861 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt
     4921 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg
     4981 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5041 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5101 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5161 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     5221 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5281 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5341 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5401 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5461 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5521 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5581 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5641 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5701 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5761 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5821 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     5881 gcactctcag tac
//
