LOCUS       sequence-337685-        7960 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 7960)
  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 7960)
  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..7960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2596..2664
                     /label=recA (Delta-LexA) promoter
                     /note="constitutively active E. coli recA promoter lacking 
                     the LexA binding site (Brent and Ptashne, 1981)"
     primer_bind     complement(3735..3752)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3904..3950
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(4077..4082,4083..4098,4099..4105)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4113..4129
                     /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)."
     rep_origin      complement(5556..6144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5636..5655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             6890..7684
                     /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             6890..7684
                     /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(6944..6963)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7554..7573
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      7697..7833
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
ORIGIN
        1 gcgatcgcgg ctcccgacat cttggaccat tagccgaact tttgctgagt tgaaggatcc
       61 tcgggttgtc agcctgtccc gcttataaga tcatacgccg ttatacgttg tttacgcttt
      121 gaggaatccc aatgatgttc aaacgcgaag aaatcatcga aatggcgaac aaagacttcg
      181 aaaaggcctg gattgaaacc aaggatctga tcaaggcgaa gaagatcaac gagagctatc
      241 cacgtatcaa accggtgttc ggcaaaaccc atccggtaaa cgacacgatc gagaacctgc
      301 gtcaggccta cctgcgtatg ggttttgaag agtatatcaa cccggtgatt gtggacgaac
      361 gcgatatcta caagcagttt ggtccagaag ccatggcagt gctggaccgt tgtttctacc
      421 tggcaggttt accacgtcct gatgtcggtc tgtctgatga aaagatcagc cagatcgaga
      481 aactggggat caaagttagc gaacacaaag agagcctgca gaagattctg cacggctaca
      541 agaaaggcac cctggatggc gatgatctgg tcctggagat tagcaacgct ctggagatca
      601 gctcggagat ggggctgaag atcctggaag atgtgtttcc ggagttcaaa gatctgacgg
      661 cagtttcctc taagctgacg ctgcgtagtc atatgaccag cggctggttt ctgaccgtgt
      721 ctgacctgat gaacaagaaa ccgttgccgt tcaaactgtt cagcatcgat cgttgctttc
      781 gtcgtgaaca gaaggaagac aaaagccatc tgatgaccta tcattctgcc agctgtgcaa
      841 ttgcgggtga aggtgtggac atcaacgacg gcaaagctat tgccgaaggt ctgctgtccc
      901 agtttggctt tacgaacttc aagttcatcc cggatgagaa aaagtcgaag tactacaccc
      961 cagaaaccca aacggaagtt tatgcgtatc atccgaaact taaggaatgg ctggaagttg
     1021 ccacgtttgg tgtgtattct ccagtggccc tcagcaagta tggtattgat gtgccggtta
     1081 tgaatttggc ttatggcgtt gagcggctag cgatgattag cggtaacttc gcagacgtcc
     1141 gtgaaatggt atacccccag ttctacgagc acaaactgaa cgaccgcaac gttgcgtcga
     1201 tggtaaagct ggacaaagtt cccgttatgg acgagatcta cgatctgacc aaagaactga
     1261 ttgaaagctg cgtcaagaac aaggatctga aaagcccatg cgaactggca attgagaaga
     1321 cgttcagctt cggtaagacc aagaagaacg tgaagatcaa catcttccca aagtttgaag
     1381 gcaaaaacct gctgggtccg agcatcctga acgagatcta cgtgtacgac ggcaacgtga
     1441 tcgggatccc agaaagcttt gacggtgtga aagaggagtt caaggacttc ctggagaaag
     1501 gcaagagcga aggtgtggca accggtatcc gctacatcga cgcgctgtgc ttcaaaatca
     1561 ccagcaagct ggaggaagcg tttgtgagca acacgaccga gttcaaggtg aaagtgatgt
     1621 gggtgcgtag cctgtccgac atcaacctga aaatcgacga tattgccctg aaacagatca
     1681 tgagcaagaa caaggtgatt gacgtgcgtg gtccagtgag tctgaacgtg gaggtgaaaa
     1741 tcgagtaatg cagtttcaaa cgctaaattg cctgatgcgc tacgcttatc aggcctacat
     1801 gatctctgca atatattgag tttgcgtgct tttgtaggcc ggataaggcg ttcacgccgc
     1861 atccggcaag aaacagcaaa caatccaaaa cgccgcgttc ggcggtcgac acagatgtag
     1921 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     1981 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgctg
     2041 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     2101 tcatgccccg cgcccaccgg aaggagctac cggcagcggt gcggactgtt gtaactcaga
     2161 ataagaaatg aggccgctca tggcgttctg ttgcccgtct cactggtgaa aagaaaaaca
     2221 accctggcgc cgcttctttg agcgaacgat caaaaataag tggcgcccca tcaaaaaaat
     2281 attctcaaca taaaaaactt tgtgtaatac ttgtaacgct agatctgtcc gcgtagtcta
     2341 ggggttaggc agcagtctct aaaattgcct tacgtgggtt caaatcccac cgcggactcc
     2401 aactagtatc cttagcgaaa gctaaggatt ttttttacat gtacgcactt tgcgccgaat
     2461 aaatacctgt gacgaagctg ttgtgaccgc ttgctctagc cagctatcga gttgtgaacc
     2521 gatccatcta gcaattggtc tcgatctagc gataggcttc gatctagcta tgtagaaacg
     2581 ccgtgtgctc gatcgcttga taaggtccac gtagctgcta taattgcttc aacagaacat
     2641 attgactatc cggtattacc cggcagatct ttgtcgatcc taccatccac tcgacacacc
     2701 cgccagcggc cgctgccaag cttccgagct ctcgaattca aaggaggtac ccaccatgcg
     2761 tgctcactac tcttacctga aaggtgacaa cgttgctgtt gctcagtgcc cggcttcttg
     2821 cggtgaactg atccagggtt ggatcctggg ttctgaaaaa ctggtttctt gcccggttga
     2881 ctggtactct accgttgctg ttaccgctgc tccgccgctg atcaacgaac gtccgctgtc
     2941 tcgtgctatg gttgaacgtg ttctggctca ctggcagtac ccggctcact ggtctaacga
     3001 aatccgtgtt gacgttcgtt cttctatccc ggttgctaaa ggtatggctt cttctaccgc
     3061 tgacatcgct gctaccgctg ttgctaccgc tcaccacctg ggtcactctc tggacgaaac
     3121 caccctggct cagctgtgcg tttctatcga accgaccgac tctaccgttt tccaccagct
     3181 gaccctgttc gaccacaaca acgctgctac ccagatcgct tgcgaaccgc cgccgccgat
     3241 cgacctgctg gttctggaat ctccggttac cctgcgtacc caggactacc accgtctgcc
     3301 gcgtcagcag aaactgatcg cttcttctgc taccctgcag caggcttgga acctggttca
     3361 ggaagcttgc atcacccaga acccgctgcg tctgggtgaa gctgctaccc tgtctgctat
     3421 cgcttctcag accctgctgc cgaaaccggg tttcaccgct ctgctgtctc tggttgaaga
     3481 atgcgacctg tacggtctga acgttgctca ctctggttct gttgttggtc tgatgctgga
     3541 ccgtaaacgt cacgacatcg ctcgtctgaa aggtaaactg gctgaaaaaa aactgacccg
     3601 tcactggccg aaacagcacc tgctgaaaat ggttaccggt ggtgttaaac tgcagtaatt
     3661 tttttaaggc agttattggt gcgtttaaac gacgtccagc ttggctgttt tggcggatga
     3721 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     3781 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     3841 aaacgccgta gcgccgatgg tagtgtgagg cctcccatgc gagagtaggg aactgccagg
     3901 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat cctgaattga
     3961 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4021 gggatctcga cgctctccct tatgcgactc ctgcattagg attctgaaat gagctgttga
     4081 caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacaagga
     4141 ggtccatgtc taaagaaaag tttgaacgta caaaaccgca cgttaacgtc ggtactatcg
     4201 gccacgttga ccatggtaaa acaacgctga ccgctgcaat cactaccgta ctggctaaaa
     4261 cctacggcgg tgctgctcgc gcattcgacc agatcgataa cgcgccggaa gaaaaagctc
     4321 gtggtatcac catcaacact tctcgcgttg aatacgacac cccgacccgt cactacgcac
     4381 acgtagactg cccggggcac gccgactatg ttaaaaacat gatcaccggt gctgcgcaga
     4441 tggacggcgc gatcctggta gttgctgcga ctgacggccc gatgccgcag actcgtgagc
     4501 acatcctgct gggtcgtcag gtaggcgttc cgtacatcat cgtgttcctg aacaaatgcg
     4561 acatggttga tgacgaagag ctgctggaac tggttgaaat ggaagttcgt gaacttctgt
     4621 ctcagtacga cttcccgggc gacgacactc cgatcgttcg tggttctgct ctgaaagcgc
     4681 tggaaggcga cgcagagtgg gaagcgaaaa tcctggaact ggctggcttc ctggattctt
     4741 acattccgga accagagcgt gcgattgaca agccgttcct gctgccgatc aacggcgtat
     4801 actccatctc cggtcgtggt accgttgtta gcggtcgtgt agaacgcggt atcatcaaag
     4861 ttggtgaaga agttgaaatc gttggtatca aagagactca gaagtctacc tgtactggcg
     4921 ttgaaatgtt ccgcaaactg ctggacgaag gccgtgctgg tgagtgggta ggtgttctgc
     4981 tgcgtggtat caaacgtgaa gaaatcgaac gtggtcaggt actggctaag ccgggcacca
     5041 tcaagccgca caccaagttc gaatctgaag tgtacattct gtccaaagat gaaggcggcc
     5101 gtcatactcc gttcttcaaa ggctaccgtc cgcagttcta cttccgtact actgacgtga
     5161 ctggtaccat cgaactgccg gaaggcgtag agatggtaat gccgggcgac aacatcaaaa
     5221 tggttgttac cctgatccac ccgatcgcga tggacgacgg tctgcgtttc gcaatccgtg
     5281 aaggcggccg taccgttggc gcgggcgttg tagcaaaagt tctgagctaa atctcattgg
     5341 ttaccttggg ctatcgaaac ttaattaacc agtcaagtca gctacttggc gagatcgact
     5401 tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg tccttgctat
     5461 tgcacccgtt ctccgattac gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag
     5521 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5581 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5641 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5701 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5761 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5821 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5881 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5941 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6001 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6061 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6121 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6181 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6241 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6301 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6361 tttcgttcat ccatagttgc atttaaattt ccgaactctc caaggccctc gtcggaaaat
     6421 cttcaaacct ttcgtccgat ccatcttgca ggctacctct cgaacgaact atcgcaagtc
     6481 tcttggccgg ccttgcgcct tggctattgc ttggcagcgc ctatcgccag gtattactcc
     6541 aatcccgaat atccgagatc gggatcaccc gagagaagtt caacctacat cctcaatccc
     6601 gatctatccg agatccgagg aatatcgaaa tcggggcgcg cctggtgtac cgagaacgat
     6661 cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca gtcggaatcc
     6721 agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt cacggcagcg
     6781 taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata atcgagtcct
     6841 agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca tgattgaaca
     6901 agatggattg cacgcaggtt ctccggcggc ttgggtggag aggctattcg gctatgactg
     6961 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     7021 tccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     7081 agcgcggcta tcgtggctgg cgacgacggg cgttccttgc gcggctgtgc tcgacgttgt
     7141 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     7201 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     7261 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     7321 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     7381 gctcgcgcca gccgaactgt tcgccaggct caaggcgtct atgcccgacg gcgaggatct
     7441 cgtcgtgacc cacggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     7501 tggattcatc gactgtggcc gtctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     7561 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc ttgtgcttta
     7621 cggtatcgcc gcgcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     7681 ctgaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt
     7741 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca
     7801 tacgtgtcct ccttaccaga aatttatcct taaggtcgtt taaactcgac tctggctcta
     7861 tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg ctcgtcgggc
     7921 atcgaatctc gtcagctatc gtcagcttac ctttttggca
//
