LOCUS       sequence-203054-        5409 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 5409)
  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 5409)
  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..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..19,20..25,26..42,43..48,49..74)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             84..95
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2634..2680
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2727..3353)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3258..3277
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(3366..3400)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     rep_origin      complement(3597..4185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3677..3696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4273..4367)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4398..5192)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRQ
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4509..4528)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5119..5138
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5371..5389
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacatcatc
       61 aggacgcact gaccgaattc attaaagagg agaaaggtac catggggagt gggtctttca
      121 ttgctgggct tgtcaacgat ggtaatacgt gttttatgaa ctcggttctt cagtcccttg
      181 ctagtagccg tgaacttatg gagtttttgg ataataatgt aatccgtaca tatgaagaaa
      241 ttgaacagaa cgagcacaat gaggaaggta atggccaaga gagcgcacaa gatgaggcaa
      301 ctcacaaaaa aaacactcgc aagggaggta aggtctatgg gaagcataaa aagaaattaa
      361 accgcaaatc ttctagcaag gaagacgaag aaaagtcgca agaaccagac attacgtttt
      421 cggtggcgtt gcgtgatctg ctgagcgcat taaatgctaa gtattatcgc gacaaaccct
      481 actttaagac taactcttta ttaaaagcga tgagcaagtc cccgcgcaaa aatatcttgc
      541 ttgggtacga tcaagaagac gctcaggaat tttttcaaaa cattcttgcg gagttagaat
      601 ctaatgtcaa gtcgttaaac acagaaaagc ttgatactac accggtagcc aagtccgaac
      661 ttccagacga tgctctggtt ggccaattaa accttggtga ggtaggcaca gtgtacattc
      721 ccacagaaca aattgacccc aattcgattt tacatgacaa atcgattcaa aactttaccc
      781 cctttaaact gatgaccccg ttggatggga tcacggctga gcgcatcggc tgcctgcaat
      841 gcggagagaa cgggggaatt cgctacagtg ttttcagcgg attaagtttg aacctgccga
      901 atgaaaatat tggaagcact cttaaactgt cccagttact gtccgattgg tcgaaacccg
      961 agattatcga gggtgttgaa tgcaaccgtt gcgctttaac agctgcgcac tcacacttgt
     1021 ttggccaatt aaaggagttt gagaagaaac ctgaaggctc gattcccgaa aaacttatta
     1081 atgccgtaaa ggaccgcgtg caccagatcg aagaggtctt ggcaaagccg gttatcgacg
     1141 atgaagatta taaaaaattg catactgcga atatggtccg caagtgttca aaaagtaaac
     1201 aaattcttat ctctcgtcca ccacctttgt tgtctattca tatcaaccgc tctgttttcg
     1261 acccgcgcac ctacatgatt cgcaagaaca actccaaggt tttgttcaag tcacgcttga
     1321 acctggcacc ctggtgctgt gatatcaacg aaatcaatct tgacgcacgc cttccgatgt
     1381 cgaagaagga aaaagcagct caacaagatt cttctgaaga cgagaacatt ggcggagagt
     1441 actatactaa attgcatgaa cgttttgagc aggagtttga agattctgaa gaagagaagg
     1501 aatacgatga tgcagagggt aattatgcat cgtattataa ccataccaag gacatctcca
     1561 actacgatcc attgaatgga gaagtcgacg gtgtgacttc cgatgatgag gatgaataca
     1621 ttgaagagac agacgcgttg gggaatacca tcaaaaaacg tattattgaa cactccgacg
     1681 tggagaacga aaacgtgaag gataatgaag aacttcagga gatcgataac gttagcttgg
     1741 atgagccaaa aattaatgtc gaggaccagc ttgaaacgag ttctgatgag gaagacgtta
     1801 ttcctgctcc acccatcaac tacgctcgca gctttagtac ggtcccagcg acccctttaa
     1861 cttactcttt gcgcagcgtc atcgtgcact atgggactca caactacgga cattatattg
     1921 catttcgcaa gtatcgtgga tgttggtggc gcatctccga tgagacggtc tatgtggtag
     1981 atgaggccga agtactgtca acaccggggg tatttatgct tttctacgag tatgatttcg
     2041 acgaggagac cggaaaaatg aaagacgact tagaagctat ccagagcaat aatgaggaag
     2101 atgacgagaa agaacaggaa cagaagggtg tccaggagcc aaaagaatcc caggagcaag
     2161 gcgaaggcga agaacaagaa gaagggcaag agcaaatgaa atttgagcgt acggaggatc
     2221 atcgcgacat ttcagggaag gatgtgaatt aataataaaa ggagatatac catgggtaaa
     2281 acgaacgact ggctggactt tgatcaactg gcggaagaaa aagttcgcga cgcgctaaaa
     2341 ccgccatcta tgtataaagt gatattagtc aatgatgatt acactccgat ggagtttgtt
     2401 attgacgtgt tacaaaaatt cttttcttat gatgtagaac gtgcaacgca attgatgctc
     2461 gctattcact accaggggaa ggccatttgc ggagtcttta ccgccgaggt tgcagaaacc
     2521 aaagtggcga tggtgaacaa gtacgcgagg gagaatgagc atccattgct gtgtacgcta
     2581 gaaaaagcct aaaagcttga tgggggatcc catggtacgc gtgctagagg catcaaataa
     2641 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     2701 tctcattttc gccagatatc gacgtcttaa gacccacttt cacatttaag ttgtttttct
     2761 aatccgcata tgatcaattc aaggccgaat aagaaggctg gctctgcacc ttggtgatca
     2821 aataattcga tagcttgtcg taataatggc ggcatactat cagtagtagg tgtttccctt
     2881 tcttctttag cgacttgatg ctcttgatct tccaatacgc aacctaaagt aaaatgcccc
     2941 acagcgctga gtgcatataa tgcattctct agtgaaaaac cttgttggca taaaaaggct
     3001 aattgatttt cgagagtttc atactgtttt tctgtaggcc gtgtacctaa atgtactttt
     3061 gctccatcgc gatgacttag taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa
     3121 tcttgccagc tttccccttc taaagggcaa aagtgagtat ggtgcctatc taacatctca
     3181 atggctaagg cgtcgagcaa agcccgctta ttttttacat gccaatacaa tgtaggctgc
     3241 tctacaccta gcttctgggc gagtttacgg gttgttaaac cttcgattcc gacctcatta
     3301 agcagctcta atgcgctgtt aatcacttta cttttatcta atctagacat cattaattcc
     3361 taattgctag cattgtacct aggactgagc tagccataaa gttgacacta cgctctcctg
     3421 agtaggacaa atccgccgcc ctagacctag ggcgttcggc tgcggcgagc ggtatcagct
     3481 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3541 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3601 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3661 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3721 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3781 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3841 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3901 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3961 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4021 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4081 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4141 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4201 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4261 actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa
     4321 atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctctc
     4381 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     4441 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     4501 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     4561 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     4621 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
     4681 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     4741 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     4801 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     4861 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     4921 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     4981 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
     5041 gtcttgacaa aaagaacctg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     5101 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     5161 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     5221 tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     5281 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccga cgtctaagaa
     5341 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     5401 cacctcgag
//
