LOCUS       sequence-334784-        4969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of truncated yeast Ash1 including encoding 
            amino acids 420-480 with phosphorylation sites 429 and 470 removed.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4969)
  AUTHORS   Martin EW, Holehouse AS, Grace CR, Hughes A, Pappu RV, Mittag T
  TITLE     Sequence Determinants of the Conformational Properties of an 
            Intrinsically Disordered Protein Prior to and upon Multisite 
            Phosphorylation.
  JOURNAL   J Am Chem Soc. 2016 Nov 30;138(47):15323-15335. doi: 
            10.1021/jacs.6b10272. Epub 2016 Nov 17.
  PUBMED    27807972
REFERENCE   2  (bases 1 to 4969)
  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/jacs.6b10272"; journalName: "J Am Chem Soc"; date: 
            "2016-11-30- 30"; volume: "138"; issue: "47"; pages: "15323-15335"
COMMENT     Sequence Label: Ash1-5p
FEATURES             Location/Qualifiers
     source          1..4969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     128..147
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     231..250
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        231..249
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             281..303
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             323..340
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    350..374
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             377..397
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(648..672)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(725..743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      739..786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1152..1157,1158..1174,1175..1180))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1207..1225)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1293..1397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1398..1466,1467..2258)
                     /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(1616..1635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2429..3017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2918..2937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3171..3188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3203..3342
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3268..3287)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3428..3450
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3444..3635)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
       61 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
      121 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
      181 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
      241 cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac tttaagaagg
      301 agatatacat atgtcgtact accatcacca tcaccatcac ctcgaatcaa caagtttgta
      361 caaaaaagca ggcttcgaaa acctgtattt tcagggcgcc tctgcatctt catctccatc
      421 tccctccgca ccgacgaaaa gtggcaagat gagatcaaga tcatcctcac ctgtgcgtcc
      481 caaggcatac actccaagcc caaggtcacc caattatcat agatttgctc tcgactctcc
      541 ccctcaagcc cctcgaagat catcaaattc aagcataacg aaaaagggaa gtagaaggtc
      601 cagtggctca tcacccactc gtcacaccac aagagtgtga gtgtgagacc cagctttctt
      661 gtacaaagtg gttgattcga ggctgctaac aaagcccgaa aggaagctga gttggctgct
      721 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      781 tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat
      841 cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg
      901 gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag
      961 cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg
     1021 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     1081 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     1141 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     1201 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1261 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1321 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1381 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1441 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1501 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1561 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1621 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1681 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1741 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1801 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1861 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1921 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1981 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2041 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2101 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2161 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2221 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2281 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2341 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2401 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2461 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2521 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2581 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2641 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2701 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2761 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2821 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2881 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2941 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3001 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3061 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3121 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3181 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3241 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3301 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3361 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3421 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3481 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3541 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3601 gccatgttaa gggcggtttt ttcctgtttg gtcactgacg cctccgtgta agggggattt
     3661 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3721 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3781 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3841 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3901 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     3961 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4021 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4081 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     4141 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     4201 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     4261 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     4321 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     4381 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     4441 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     4501 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     4561 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     4621 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4681 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4741 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4801 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4861 agctgactgg gttgaaggct ctcaagggca tcggtcgatc gacgctctcc cttatgcgac
     4921 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccg
//
