LOCUS       sequence-307840-        8181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sfGFP-TetR fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8181)
  AUTHORS   Gibson BA, Doolittle LK, Schneider MWG, Jensen LE, Gamarra N, Henry 
            L, Gerlich DW, Redding S, Rosen MK
  TITLE     Organization of Chromatin by Intrinsic and Regulated Phase 
            Separation.
  JOURNAL   Cell. 2019 Oct 3;179(2):470-484.e21. doi: 
            10.1016/j.cell.2019.08.037. Epub 2019 Sep 19.
  PUBMED    31543265
REFERENCE   2  (bases 1 to 8181)
  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: 
            "10.1016/j.cell.2019.08.037"; journalName: "Cell"; date: "2019-10-3-
            3"; volume: "179"; issue: "2"; pages: "470-484.e21"
COMMENT     Sequence Label: pETduet+sfGFP-TetR
FEATURES             Location/Qualifiers
     source          1..8181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        89..107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    108..132
                     /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             147..169
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             182..208
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     CDS             209..229
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             233..946
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(263..291)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     886..906
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        1696..1714
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1715..1739
                     /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)."
     CDS             3235..3279
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3317..3335)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3331..3378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3415..3870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3502..3521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3712..3733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3985..4776,4777..4845))
                     /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     4608..4627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4846..4938)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5019..5607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5508..5527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5761..5778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5793..5932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5858..5877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6018..6040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6034..6225)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6797..7879)
                     /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     7841..7860
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7880..7957)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(8163..8181,1)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
       61 atcgagatcg atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      121 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      181 acatcaccat catcaccacc atcatcacga aaacctgtat tttcagggat ccatgagtaa
      241 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
      301 tgggcacaaa ttttctgtcc gtggagaggg tgaaggtgat gcaacaaacg gaaaacttac
      361 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
      421 tttaacttat ggtgttcaat gcttttcaag atacccagat catatgaaac agcatgactt
      481 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatctt tcaaagatga
      541 cgggacctac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
      601 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
      661 caactttaac tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc
      721 taacttcaaa attagacaca acgttgaaga tggaagcgtt caactagcag accattatca
      781 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
      841 acaatctgtc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
      901 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaaggcg ggtccggtgg
      961 aagtggcggg tccgcacggc tgaacagaga atcggttatt gatgcggcac tggaactgct
     1021 gaatgagaca gggattgacg ggctgacgac ccgcaagctg gcgcagaagc tgggaataga
     1081 acagccgaca ctttactggc atgtgaaaaa taaacgggcg ttactggatg cgctggcggt
     1141 ggagatcttg gcgcgtcatc atgattattc actgcctgcg gcgggggaat cttggcagtc
     1201 atttctgcgc aataatgcaa tgagtttccg ccgggcgctg ctgcgttacc gtgacggggc
     1261 aaaagtgcac ctcggcaccc gccctgatga aaaacagtat gatacggtgg aaacccagtt
     1321 acgctttatg acagaaaacg gcttttcact gcgcgacggg ttatatgcga tttcagcggt
     1381 cagtcatttt acccttggtg ccgtactgga gcagcaggag catactgccg ccctgaccga
     1441 ccgccctgca gcaccggacg aaaacctgcc gccgctattg cgggaagcgc tgcagattat
     1501 ggacagtgat gatggtgagc aggcctttct gcatggcctg gagagcctga tccgggggtt
     1561 tgaggtgcag cttacggcac tgttgcaaat agtcggtggt gataaactta tcatcccctt
     1621 ttgctgataa gcggccgcat aatgcttaag tcgaacagaa agtaatcgta ttgtacacgg
     1681 ccgcataatc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     1741 catcttagta tattagttaa gtataagaag gagatataca tagttcatat ggaccccttg
     1801 gcagtgtcag cagcttcagt agtgtcaatg tccaatgacg ttttgaaacc agagacgccc
     1861 aaggggccaa tcataatcag taaaaaccca tcaaatggta ttttctatgg tccctccttc
     1921 actaaacgag agtctctcaa tgctcgaatg tttctgaaat actatggtgc acacaaattt
     1981 ttagacactt acctccccga ggatttgaac tcgttataca tttactatct tatcaagttg
     2041 ctaggctttg aagttaaaga tcaagcgctt atcggcacca tcaacagtat tgtccatatc
     2101 aactcgcaag agcgtgttca agatttggga agtgcaatat ctgtcacaaa tgttgaagac
     2161 ccattggcaa aaaagcaaac agttcgtcta atcaaagatt tgcaaagagc aattaacaaa
     2221 gttctatgta caagattaag attatccaat tttttcacta ttgatcattt tattcgaaaa
     2281 ttacataccg ctagaaaaat tttggtcctg actggtgcag gtgtttcaac ttcattaggc
     2341 atcccggact tcagatcttc tgaggggttc tattcaaaga tcaaacattt ggggctcgat
     2401 gatccccaag acgttttcaa ttacaatata tttatgcacg acccctctgt tttctataat
     2461 attgccaata tggttttacc tccagaaaaa atttattctc cattgcatag tttcattaag
     2521 atgctacaaa tgaaagggaa attattgaga aattatactc aaaacattga taatttggaa
     2581 tcttatgcgg gaataagcac agataaactg gtgcagtgcc atggctcttt tgctactgcc
     2641 acctgcgtta cctgccattg gaacctaccc ggtgagagga tatttaataa aattagaaac
     2701 ctcgaacttc cactatgccc gtactgttac aaaaaaagaa gagaatattt cccagaggga
     2761 tataataata aagtaggtgt tgctgcatca cagggttcaa tgtcggaaag gcctccatat
     2821 atccttaact catatggcgt tctcaaacca gatatcacat tctttggcga agcactgcca
     2881 aataaatttc ataagagcat tcgcgaagat atcttagaat gtgatttgtt gatttgcatt
     2941 gggacaagtt taaaagtagc gccagtgtct gaaatcgtaa acatggttcc ttcccacgtt
     3001 ccccaagtcc tgattaatcg tgatcccgtc aagcacgcag aatttgattt atctcttttg
     3061 gggtactgtg atgacattgc agctatggta gcccaaaaat gtggctggac gattccgcat
     3121 aagaaatgga acgatttgaa gaacaagaac tttaaatgcc aagagaagga taagggcgtg
     3181 tatgtcgtta catcagatga acatcccaaa accctctaat gactcgagtc tggtaaagaa
     3241 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     3301 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     3361 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     3421 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3481 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3541 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3601 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3661 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3721 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3781 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3841 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     3901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4441 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4501 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4561 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4621 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4681 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4741 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4801 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4861 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4921 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4981 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5041 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5101 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5161 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5221 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5281 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5341 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5401 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5461 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5521 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5581 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5641 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5701 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5761 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5821 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5881 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5941 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6001 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6061 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     6121 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     6181 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     6241 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     6301 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     6361 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     6421 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     6481 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6541 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6601 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6661 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     6721 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     6781 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6841 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     6901 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6961 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     7021 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     7081 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     7141 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     7201 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     7261 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     7321 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     7381 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     7441 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     7501 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     7561 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     7621 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     7681 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     7741 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     7801 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     7861 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     7921 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     7981 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     8041 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     8101 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     8161 tcggtgatgt cggcgatata g
//
