LOCUS       sequence-460719-        5569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of N-terminally 6His tagged FL Gcn4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5569)
  AUTHORS   Bremer A, Lang WH, Kempen RP, Sweta K, Taylor AB, Borgia MB, Ansari 
            AZ, Mittag T
  TITLE     Reconciling competing models on the roles of condensates and soluble
            complexes in transcription factor function.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 22:2024.11.21.624739. doi: 
            10.1101/2024.11.21.624739.
  PUBMED    39605529
REFERENCE   2  (bases 1 to 5569)
  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"; journalName: "bioRxiv 
            [Preprint]. 2024 Nov 22:2024.11.21.624739. doi: 
            10.1101/2024.11.21.624739."
COMMENT     Sequence Label: pDest17_FL Gcn4
FEATURES             Location/Qualifiers
     source          1..5569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             71..93
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             113..130
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    140..164
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             167..187
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(1038..1062)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1115..1133)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1129..1176
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1542..1547,1548..1564,1565..1570))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1597..1615)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1683..1787
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1788..1856,1857..2648)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2006..2025)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2819..3407
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3308..3327
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3561..3578
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3593..3732
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3658..3677)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3818..3840
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3834..4025)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5487..5506
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
       61 tagaaataat tttgtttaac tttaagaagg agatatacat atgtcgtact accatcacca
      121 tcaccatcac ctcgaatcaa caagtttgta caaaaaagca ggcttcgaga acctgtattt
      181 tcagggtagc atgagcgaat atcagccgag cctgtttgca ctgaatccga tgggttttag
      241 tccgctggat ggtagcaaaa gcaccaatga aaatgttagc gcaagcacca gcaccgcaaa
      301 accgatggtt ggtcagctga tttttgacaa attcatcaaa accgaagagg atccgatcat
      361 caaacaggat accccgagca atctggattt tgattttgca ctgccgcaga ccgcaaccgc
      421 accggatgca aaaaccgttc tgccgattcc ggaactggat gatgcagttg ttgaaagctt
      481 ttttagcagc agcaccgata gcaccccgat gtttgaatat gaaaatctgg aagataacag
      541 caaagaatgg accagcctgt tcgataatga tattccggtt accaccgatg atgttagcct
      601 ggcagataaa gcaattgaaa gcaccgaaga agtgagcctg gttccgagta acctggaagt
      661 tagcaccacc agttttctgc cgacaccggt gctggaagat gcaaaactga cccagacacg
      721 taaagtgaaa aaaccgaata gcgtggtgaa aaaaagccat catgttggca aagatgatga
      781 aagccgtctg gatcatctgg gtgttgttgc atataatcgt aaacagcgta gcattccgct
      841 gagcccgatt gttccggaaa gcagcgatcc ggcagcactg aaacgtgccc gtaataccga
      901 agcagcacgt cgtagccgtg cacgtaaact gcagcgtatg aaacagttag aagataaggt
      961 tgaagaactg ctgtgcaaaa actatcacct ggaaaatgaa gttgcccgtc tgaaaaaact
     1021 ggttggcgaa cgttaagacc cagctttctt gtacaaagtg gttgattcga ggctgctaac
     1081 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1141 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1201 tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc caagtagcga
     1261 agcgagcagg actgggcggc ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca
     1321 tagaaattgc atcaacgcat atagcgctag cagcacgcca tagtgactgg cgatgctgtc
     1381 ggaatggacg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
     1441 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
     1501 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
     1561 aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     1621 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     1681 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1741 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1801 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1861 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1921 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1981 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     2041 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2101 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2161 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2221 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2281 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2341 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2401 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2461 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2521 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2581 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2641 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2701 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2761 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2821 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2881 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2941 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3001 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3061 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3121 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3181 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3241 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3301 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3361 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3421 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3481 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3541 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3601 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3661 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3721 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3781 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3841 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3901 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3961 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4021 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4081 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4141 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4201 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4261 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4321 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4381 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4441 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     4501 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     4561 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     4621 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     4681 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     4741 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     4801 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     4861 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     4921 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     4981 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     5041 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     5101 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     5161 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5221 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5281 tcggtcgatc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     5341 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5401 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     5461 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     5521 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcg
//
