LOCUS       sequence-352368-        7668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The pThr/tRNAv2.0CUA pair enables the biosynthesis of proteins 
            containing pThr.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7668)
  AUTHORS   Zhang MS, Brunner SF, Huguenin-Dezot N, Liang AD, Schmied WH, 
            Rogerson DT, Chin JW
  TITLE     Biosynthesis and genetic encoding of phosphothreonine through 
            parallel selection and deep sequencing.
  JOURNAL   Nat Methods. 2017 Jul;14(7):729-736. doi: 10.1038/nmeth.4302. Epub 
            2017 May 29.
  PUBMED    28553966
REFERENCE   2  (bases 1 to 7668)
  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/nmeth.4302"; journalName: "Nat Methods"; date: "2017-07"; 
            volume: "14"; issue: "7"; pages: "729-736"
COMMENT     Sequence Label: pUC_pThrRS_SeptRNAv2.0CUA_OXB20-PduX_EFSep
FEATURES             Location/Qualifiers
     source          1..7668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(100..688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(180..199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             1434..2228
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1434..2228
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1488..1507)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2098..2117
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2241..2377
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     promoter        5100..5168
                     /label=recA (Delta-LexA) promoter
                     /note="constitutively active E. coli recA promoter lacking 
                     the LexA binding site (Brent and Ptashne, 1981)"
     promoter        join(6289..6294,6295..6310,6311..6317)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6325..6341
                     /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)."
ORIGIN
        1 ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca
       61 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      421 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      481 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      541 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      601 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
      661 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
      721 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
      781 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
      841 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
      901 tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga
      961 aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca
     1021 agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta
     1081 ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa
     1141 tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggt gtaccgagaa
     1201 cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga
     1261 atccagcttg ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc
     1321 agcgtaccga tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag
     1381 tcctagcttt tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgattg
     1441 aacaagatgg attgcacgca ggttctccgg cggcttgggt ggagaggcta ttcggctatg
     1501 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     1561 ggcgtccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     1621 aggcagcgcg gctatcgtgg ctggcgacga cgggcgttcc ttgcgcggct gtgctcgacg
     1681 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     1741 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     1801 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     1861 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     1921 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gtctatgccc gacggcgagg
     1981 atctcgtcgt gacccacggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     2041 tttctggatt catcgactgt ggccgtctgg gtgtggcgga ccgctatcag gacatagcgt
     2101 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttccttgtgc
     2161 tttacggtat cgccgcgccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     2221 tcttctgacc gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg
     2281 tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact
     2341 tccatacgtg tcctccttac cagaaattta tccttaaggt cgtttaaact cgactctggc
     2401 tctatcgaat ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc
     2461 gggcatcgaa tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg
     2521 acatcttgga ccattagccg aacttttgct gagttgaagg atcctcgggt tgtcagcctg
     2581 tcccgcttat aagatcatac gccgttatac gttgtttacg ctttgaggaa tcccaatgat
     2641 gttcaaacgc gaagaaatca tcgaaatggc gaacaaagac ttcgaaaagg cctggattga
     2701 aaccaaggat ctgatcaagg cgaagaagat caacgagagc tatccacgta tcaaaccggt
     2761 gttcggcaaa acccatccgg taaacgacac gatcgagaac ctgcgtcagg cctacctgcg
     2821 tatgggtttt gaagagtata tcaacccggt gattgtggac gaacgcgata tctacaagca
     2881 gtttggtcca gaagccatgg cagtgctgga ccgttgtttc tacctggcag gtttaccacg
     2941 tcctgatgtc ggtctgtctg atgaaaagat cagccagatc gagaaactgg ggatcaaagt
     3001 tagcgaacac aaagagagcc tgcagaagat tctgcacggc tacaagaaag gcaccctgga
     3061 tggcgatgat ctggtcctgg agattagcaa cgctctggag atcagctcgg agatggggct
     3121 gaagatcctg gaagatgtgt ttccggagtt caaagatctg acggcagttt cctctaagct
     3181 gacgctgcgt agtcatatga ccagcggctg gtttctgacc gtgtctgacc tgatgaacaa
     3241 gaaaccgttg ccgttcaaac tgttcagcat cgatcgttgc tttcgtcgtg aacagaagga
     3301 agacaaaagc catctgatga cctatcattc tgccagctgt gcaattgcgg gtgaaggtgt
     3361 ggacatcaac gacggcaaag ctattgccga aggtctgctg tcccagtttg gctttacgaa
     3421 cttcaagttc atcccggatg agaaaaagtc gaagtactac accccagaaa cccaaacgga
     3481 agtttatgcg tatcatccga aacttaagga atggctggaa gttgccacat ttggtgtgta
     3541 ttctccagtg gccctcagca agtatggtat tgatgtgccg gttatgaatt tggcttatgg
     3601 cgttgagcgg ctagcgatga ttagcggtaa cttcgcagac gtccgtgaaa tggtataccc
     3661 ccagttctac gagcacaaac tgaacgaccg caacgttgcg tcgatggtaa agctggacaa
     3721 agttcccgtt atggacgaga tctacgatct gaccaaagaa ctgattgaaa gctgcgtcaa
     3781 gaacaaggat ctgaaaagcc catgcgaact ggcaattgag aagacgttca gcttcggtaa
     3841 gaccaagaag aacgtgaaga tcaacatctt cccaaagttt gaaggcaaaa acctgctggg
     3901 tccgagcatc ctgaacgaga tctacgtgta cgacggcaac gtgatcggga tcccagaaag
     3961 ctttgacggt gtgaaagagg agttcaagga cttcctggag aaaggcaaga gcgaaggtgt
     4021 ggcaaccggt atccgctaca tcgacgcgct gtgcttcaaa atcaccagca agctggagga
     4081 agcgtttgtg agcaacacga ccgagttcaa ggtgaaagtg atgtgggtgc gtagcctgtc
     4141 cgacatcaac ctgaaaatcg acgatattgc cctgaaacag atcatgagca agaacaaggt
     4201 gattgacgtg cgtggtccag tgagtctgaa cgtggaggtg aaaatcgagt aatgcagttt
     4261 caaacgctaa attgcctgat gcgctacgct tatcaggcct acatgatctc tgcaatatat
     4321 tgagtttgcg tgcttttgta ggccggataa ggcgttcacg ccgcatccgg caagaaacag
     4381 caaacaatcc aaaacgccgc gttcggcggt cgacacagat gtaggtgttc cacagggtag
     4441 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctt gtttcggcgt
     4501 gggtatggtg gcaggccccg tggccggggg actgttgggc gctgccggca cctgtcctac
     4561 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4621 ccggaaggag ctaccggcag cggtgcggac tgttgtaact cagaataaga aatgaggccg
     4681 ctcatggcgt tctgttgccc gtctcactgg tgaaaagaaa aacaaccctg gcgccgcttc
     4741 tttgagcgaa cgatcaaaaa taagtggcgc cccatcaaaa aaatattctc aacataaaaa
     4801 actttgtgta atacttgtaa cgctagatct gtccgcgtag tctaggggtt aggcagcagt
     4861 ctctaaaatt gccttacgtg ggttcaaatc ccaccgcgga ctccaactag tatccttagc
     4921 gaaagctaag gatttttttt acatgtacgc actttgcgcc gaataaatac ctgtgacgaa
     4981 gctgttgtga ccgcttgctc tagccagcta tcgagttgtg aaccgatcca tctagcaatt
     5041 ggtctcgatc tagcgatagg cttcgatcta gctatgtaga aacgccgtgt gctcgatcgc
     5101 ttgataaggt ccacgtagct gctataattg cttcaacaga acatattgac tatccggtat
     5161 tacccggcag atctttgtcg atcctaccat ccactcgaca cacccgccag cggccgctgc
     5221 caagcttccg agctctcgaa ttcaaaggag gtacccacca tgcgtgctca ctactcttac
     5281 ctgaaaggtg acaacgttgc tgttgctcag tgcccggctt cttgcggtga actgatccag
     5341 ggttggatcc tgggttctga aaaactggtt tcttgcccgg ttgactggta ctctaccgtt
     5401 gctgttaccg ctgctccgcc gctgatcaac gaacgtccgc tgtctcgtgc tatggttgaa
     5461 cgtgttctgg ctcactggca gtacccggct cactggtcta acgaaatccg tgttgacgtt
     5521 cgttcttcta tcccggttgc taaaggtatg gcttcttcta ccgctgacat cgctgctacc
     5581 gctgttgcta ccgctcacca cctgggtcac tctctggacg aaaccaccct ggctcagctg
     5641 tgcgtttcta tcgaaccgac cgactctacc gttttccacc agctgaccct gttcgaccac
     5701 aacaacgctg ctacccagat cgcttgcgaa ccgccgccgc cgatcgacct gctggttctg
     5761 gaatctccgg ttaccctgcg tacccaggac taccaccgtc tgccgcgtca gcagaaactg
     5821 atcgcttctt ctgctaccct gcagcaggct tggaacctgg ttcaggaagc ttgcatcacc
     5881 cagaacccgc tgcgtctggg tgaagctgct accctgtctg ctatcgcttc tcagaccctg
     5941 ctgccgaaac cgggtttcac cgctctgctg tctctggttg aagaatgcga cctgtacggt
     6001 ctgaacgttg ctcactctgg ttctgttgtt ggtctgatgc tggaccgtaa acgtcacgac
     6061 atcgctcgtc tgaaaggtaa actggctgaa aaaaaactga cccgtcactg gccgaaacag
     6121 cacctgctga aaatggttac cggtggtgtt aaactgcagt aacctgaatt gactctcttc
     6181 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6241 gacgctctcc cttatgcgac tcctgcatta ggattctgaa atgagctgtt gacaattaat
     6301 catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaag gaggtccatg
     6361 tctaaagaaa agtttgaacg tacaaaaccg cacgttaacg tcggtactat cggccacgtt
     6421 gaccatggta aaacaacgct gaccgctgca atcactaccg tactggctaa aacctacggc
     6481 ggtgctgctc gcgcattcga ccagatcgat aacgcgccgg aagaaaaagc tcgtggtatc
     6541 accatcaaca cttctcgcgt tgaatacgac accccgaccc gtcactacgc acacgtagac
     6601 tgcccggggc acgccgacta tgttaaaaac atgatcaccg gtgctgcgca gatggacggc
     6661 gcgatcctgg tagttgctgc gactgacggc ccgatgccgc agactcgtga gcacatcctg
     6721 ctgggtcgtc aggtaggcgt tccgtacatc atcgtgttcc tgaacaaatg cgacatggtt
     6781 gatgacgaag agctgctgga actggttgaa atggaagttc gtgaacttct gtctcagtac
     6841 gacttcccgg gcgacgacac tccgatcgtt cgtggttctg ctctgaaagc gctggaaggc
     6901 gacgcagagt gggaagcgaa aatcctggaa ctggctggct tcctggattc ttacattccg
     6961 gaaccagagc gtgcgattga caagccgttc ctgctgccga tcaacggcgt atactccatc
     7021 tccggtcgtg gtaccgttgt tagcggtcgt gtagaacgcg gtatcatcaa agttggtgaa
     7081 gaagttgaaa tcgttggtat caaagagact cagaagtcta cctgtactgg cgttgaaatg
     7141 ttccgcaaac tgctggacga aggccgtgct ggtgagtggg taggtgttct gctgcgtggt
     7201 atcaaacgtg aagaaatcga acgtggtcag gtactggcta agccgggcac catcaagccg
     7261 cacaccaagt tcgaatctga agtgtacatt ctgtccaaag atgaaggcgg ccgtcatact
     7321 ccgttcttca aaggctaccg tccgcagttc tacttccgta ctactgacgt gactggtacc
     7381 atcgaactgc cggaaggcgt agagatggta atgccgggcg acaacatcaa aatggttgtt
     7441 accctgatcc acccgatcgc gatggacgac ggtctgcgtt tcgcaatccg tgaaggcggc
     7501 cgtaccgttg gcgcgggcgt tgtagcaaaa gttctgagct aaatctcatt ggttaccttg
     7561 ggctatcgaa acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg
     7621 tttcgactac gctcagaatt gcgtcagtca agttcgatct ggtccttg
//
