LOCUS       sequence-200686-        4696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses truncated codon-opt S. cerevisiae UBP1 and the V65I 
            engineered variant of E. coli ClpS in an artificial operon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4696)
  AUTHORS   Kunjapur AM, Stork DA, Kuru E, Vargas-Rodriguez O, Landon M, Soll D,
            Church GM
  TITLE     Engineering posttranslational proofreading to discriminate 
            nonstandard amino acids.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Jan 16;115(3):619-624. doi: 
            10.1073/pnas.1715137115. Epub 2018 Jan 4.
  PUBMED    29301968
REFERENCE   2  (bases 1 to 4696)
  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: 
            "10.1073/pnas.1715137115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-01-16- 16"; volume: "115"; issue: "3"; pages: "619-624"
COMMENT     Sequence Label: pZE21/UBP1/ClpS_V65I
FEATURES             Location/Qualifiers
     source          1..4696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2245..2331
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(2495..3083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2575..2594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3171..3265)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3296..4090)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3407..3426)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4017..4036
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4269..4287
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(4308..4326,4327..4332,4333..4349,4350..4355,
                     4356..4381)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    4308..4326
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4333..4351
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             4391..4402
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 gaaaagtcgc aagaaccaga cattacgttt tcggtggcgt tgcgtgatct gctgagcgca
       61 ttaaatgcta agtattatcg cgacaaaccc tactttaaga ctaactcttt attaaaagcg
      121 atgagcaagt ccccgcgcaa aaatatcttg cttgggtacg atcaagaaga cgctcaggaa
      181 ttttttcaaa acattcttgc ggagttagaa tctaatgtca agtcgttaaa cacagaaaag
      241 cttgatacta caccggtagc caagtccgaa cttccagacg atgctctggt tggccaatta
      301 aaccttggtg aggtaggcac cgtgtacatt cccacagaac aaattgaccc caattcgatt
      361 ttacatgaca aatcgattca aaactttacc ccctttaaac tgatgacccc gttggatggg
      421 atcacggctg agcgcatcgg ctgcctgcaa tgcggagaga acgggggaat tcgctacagt
      481 gttttcagcg gattaagttt gaacctgccg aatgaaaata ttggaagcac tcttaaactg
      541 tcccagttac tgtccgattg gtcgaaaccc gagattatcg agggtgttga atgcaaccgt
      601 tgcgctttaa cagctgcgca ctcacacttg tttggccaat taaaggagtt tgagaagaaa
      661 cctgaaggct cgattcccga aaaacttatt aatgccgtaa aggaccgcgt gcaccagatc
      721 gaagaggtct tggcaaagcc ggttatcgac gatgaagatt ataaaaaatt gcatactgcg
      781 aatatggtcc gcaagtgttc aaaaagtaaa caaattctta tctctcgtcc accacctttg
      841 ttgtctattc atatcaaccg ctctgttttc gacccgcgca cctacatgat tcgcaagaac
      901 aactccaagg ttttgttcaa gtcacgcttg aacctggcac cctggtgctg tgatatcaac
      961 gaaatcaatc ttgacgcacg ccttccgatg tcgaagaagg aaaaagcagc tcaacaagat
     1021 tcttctgaag acgagaacat tggcggagag tactatacta aattgcatga acgttttgag
     1081 caggagtttg aagattctga agaagagaag gaatacgatg atgcagaggg taattatgca
     1141 tcgcattata accataccaa ggacatctcc aactacgatc cattgaatgg agaagtcgac
     1201 ggtgtgactt ccgatgatga ggatgaatac attgaagaga cagacgcgtt ggggaatacc
     1261 atcaaaaaac gtattattga acactccgac gtggagaacg aaaacgtgaa ggataatgaa
     1321 gaacttcagg agatcgataa cgttagcttg gatgagccaa aaattaatgt cgaggaccag
     1381 cttgaaacga gttctgatga ggaagacgtt attcctgctc cacccatcaa ctacgctcgc
     1441 agctttagta cggtcccagc gaccccttta acttactctt tgcgcagcgt catcgtgcac
     1501 tatgggactc acaactacgg acattatatt gcatttcgca agtatcgtgg atgttggtgg
     1561 cgcatctccg atgagacggt ctatgtggta gatgaggccg aagtactgtc aacaccgggg
     1621 gtatttatgc ttttctacga gtatgatttc gacgaggaga ccggaaaaat gaaagacgac
     1681 ttagaagcta tccagagcaa taatgaggaa gatgacgaga aagaacagga acagaagggt
     1741 gtccaggagc caaaagaatc ccaggagcaa ggcgaaggcg aagaacaaga agaagggcaa
     1801 gagcaaatga aatttgagcg tacggaggat catcgcgaca tttcagggaa ggatgtgaat
     1861 taataataaa aggagatata ccatgggtaa aacgaacgac tggctggact ttgatcaact
     1921 ggcggaagaa aaagttcgcg acgcgctaaa accgccatct atgtataaag tgatattagt
     1981 caatgatgat tacactccga tggagtttgt tattgacgtg ttacaaaaat tcttttctta
     2041 tgatgtagaa cgtgcaacgc aattgatgct cgctattcac taccagggga aggccatttg
     2101 cggagtcttt accgccgagg ttgcagaaac caaagtggcg atggtgaaca agtacgcgag
     2161 ggagaatgag catccattgc tgtgtacgct agaaaaagcc taaaagcttg atgggggatc
     2221 ccatggtacg cgtgctagag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     2281 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccgccc
     2341 tagacctagg cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2401 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     2701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     2941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3121 agtggaacga aaactcacgt taagggattt tggtcatgac tagtgcttgg attctcacca
     3181 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc
     3241 attactggat ctatcaacag gagtccaagc gagctctcga accccagagt cccgctcaga
     3301 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     3361 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     3421 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     3481 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     3541 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     3601 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     3661 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     3721 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     3781 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     3841 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     3901 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     3961 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     4021 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     4081 gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatcttgat cccctgcgcc
     4141 atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac
     4201 cagagggcgc cccagctggc aattccgacg tctaagaaac cattattatc atgacattaa
     4261 cctataaaaa taggcgtatc acgaggccct ttcgtcttca cctcgagtcc ctatcagtga
     4321 tagagattga catccctatc agtgatagag atactgagca catcagcagg acgcactgac
     4381 cgaattcatt aaagaggaga aaggtaccat ggggagtggg tctttcattg ctgggcttgt
     4441 caacgatggt aatacgtgtt ttatgaactc ggttcttcag tcccttgcta gtagccgtga
     4501 acttatggag tttttggata ataatgtaat ccgtacatat gaagaaattg aacagaacga
     4561 gcacaatgag gaaggtaatg gccaagagag cgcacaagat gaggcaactc acaaaaaaaa
     4621 cactcgcaag ggaggtaagg tctatgggaa gcataaaaag aaattaaacc gcaaatcttc
     4681 tagcaaggaa gacgaa
//
