LOCUS       sequence-345707-        4650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression and purification, combined with SAE2 to form 
            the E1 enzyme heterodimer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4650)
  AUTHORS   Peeples W, Rosen MK
  TITLE     Mechanistic dissection of increased enzymatic rate in a 
            phase-separated compartment.
  JOURNAL   Nat Chem Biol. 2021 Jun;17(6):693-702. doi: 
            10.1038/s41589-021-00801-x. Epub 2021 May 25.
  PUBMED    34035521
REFERENCE   2  (bases 1 to 4650)
  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: 
            "10.1038/s41589-021-00801-x"; journalName: "Nat Chem Biol"; date: 
            "2021-06"; volume: "17"; issue: "6"; pages: "693-702"
COMMENT     Sequence Label: SAE1
FEATURES             Location/Qualifiers
     source          1..4650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(260..1342)
                     /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     1304..1323
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1343..1420)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1466..1485
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1466..1484
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1485..1509
                     /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             1559..1576
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1586..1606
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2717..2761
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2799..2817)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2813..2860
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3031..3822)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3823..3914)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(3962..4650,1..50))
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctactg
       61 aaccgctcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
      121 tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac
      181 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
      241 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
      301 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
      361 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
      421 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
      481 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
      541 tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
      601 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
      661 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
      721 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
      781 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
      841 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
      901 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
      961 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     1021 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     1081 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     1141 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     1201 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     1261 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     1321 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     1381 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     1441 ttatgcgact cctgcattag gaaattaata cgactcacta taggggaatt gtgagcggat
     1501 aacaattccc ctgtagaaat aattttgttt aactttaata aggagatata ccatgaaaca
     1561 tcaccaccac catcacggtg gatctgaaaa cctgtacttt cagggcgaat tcggaggtag
     1621 tggcggatca ggtgggagtg gagggtctat ggtggagaag gaggaggctg gcggcggcat
     1681 tagcgaggag gaggcggcac agtatgaccg gcagatccgc ctgtggggac tggaggccca
     1741 gaaacggctg cgggcctctc gggtgcttct tgtcggcttg aaaggacttg gggctgaaat
     1801 tgccaagaat ctcatcttgg caggagtgaa aggactgacc atgctggatc acgaacaggt
     1861 aactccagaa gatcccggag ctcagttctt gattcgtact gggtctgttg gccgaaatag
     1921 ggctgaagcc tctttggagc gagctcagaa tctcaacccc atggtggatg tgaaggtgga
     1981 cactgaggat atagagaaga aaccagagtc atttttcact caattcgatg ctgtgtgtct
     2041 gacttgctgc tccagggatg tcatagttaa agttgaccag atctgtcaca aaaatagcat
     2101 caagttcttt acaggagatg tttttggcta ccatggatac acatttgcca atctaggaga
     2161 gcatgagttt gtagaggaga aaactaaagt tgccaaagtt agccaaggag tagaagatgg
     2221 gcccgacacc aagagagcaa aacttgattc ttctgagaca acgatggtca aaaagaaggt
     2281 ggtcttctgc cctgttaaag aagccctgga ggtggactgg agcagtgaga aagcaaaggc
     2341 tgctctgaag cgcacgacct ccgactactt tctccttcaa gtgctcttaa agttccgtac
     2401 agataaagga agagatccca gttctgatac atatgaggaa gattctgagt tgttgctcca
     2461 gatacgaaat gatgtgcttg actcactggg tattagtcct gacctgcttc ctgaggactt
     2521 tgtcaggtac tgcttctccg agatggcccc agtgtgtgcg gtggttggag ggattttggc
     2581 acaggaaatt gtgaaggccc tgtctcagcg ggaccctcct cacaacaact tcttcttctt
     2641 cgatggcatg aaggggaatg ggattgtgga gtgccttggc cccaagtgaa agcttgcggc
     2701 cgcactcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc
     2761 gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata
     2821 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga
     2881 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
     2941 agcggctatt taacgaccct gccctgaacc gacgaccggg tcatcgtggc cggatcttgc
     3001 ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc
     3061 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
     3121 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     3181 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     3241 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     3301 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     3361 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     3421 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     3481 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     3541 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     3601 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     3661 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     3721 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     3781 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata
     3841 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3901 gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg
     3961 ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg ggactaacat
     4021 gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc
     4081 agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat
     4141 gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg
     4201 tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct
     4261 ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg
     4321 tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct
     4381 gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac
     4441 tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt
     4501 gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct
     4561 ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct tcgatcaaac
     4621 cacctcccca ggtggttttt tcgtttacag
//
