LOCUS       sequence-465531-        5062 bp DNA     circular SYN 17-MAR-2025
DEFINITION  Bacterial expression of N-terminally 6His tagged Gcn4 E2K2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5062)
  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 2024.11.21.624739
REFERENCE   2  (bases 1 to 5062)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 17, 2025 from SnapGene Server 7.0.3
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.11.21.624739"
FEATURES             Location/Qualifiers
     source          1..5062
                     /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(531..555)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(608..626)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      622..669
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(1035..1063)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1090..1108)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1176..1280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1281..2141
                     /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(1499..1518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2312..2900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2801..2820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3054..3071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3086..3225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3151..3170)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3311..3333
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3327..3518)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4980..4999
                     /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 agcaccgata aaaccccgat gtttgaatat gaaaaccttg aagataacga
      241 aaaagagtgg accagcctgt ttgataatga tattccggtt accaccgatg atgttgaact
      301 ggcagataaa gcaattgaaa aaggtagcgg tagcggttca ggtagtccgg aaagcagcga
      361 tccggcagca ctgaaacgtg cccgtaatac cgaagcagca cgtcgtagcc gtgcacgtaa
      421 actgcagcgt atgaaacagc tggaagataa agttgaagaa ctgctgtgca aaaactacca
      481 cctggaaaat gaagttgcac gtctgaaaaa gctggttggt gaacgttaag acccagcttt
      541 cttgtacaaa gtggttgatt cgaggctgct aacaaagccc gaaaggaagc tgagttggct
      601 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      661 ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat
      721 gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcggccaaa
      781 gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg catatagcgc
      841 tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc
      901 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
      961 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     1021 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     1081 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1141 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1201 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1261 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1321 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1381 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1441 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1501 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     1561 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1621 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1681 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     1741 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     1801 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     1861 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     1921 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     1981 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2041 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2101 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2161 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2221 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2281 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2341 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2401 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2461 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2521 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2581 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2641 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2701 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2761 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2821 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2881 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2941 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3001 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3061 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3121 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3181 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3241 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3301 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3361 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3421 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3481 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3541 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3601 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3661 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3721 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3781 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3841 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3901 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     3961 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     4021 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     4081 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     4141 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     4201 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     4261 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     4321 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     4381 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     4441 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     4501 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4561 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4621 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4681 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4741 aggagctgac tgggttgaag gctctcaagg gcatcggtcg atcgacgctc tcccttatgc
     4801 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     4861 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     4921 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     4981 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5041 atgcgtccgg cgtagaggat cg
//
