LOCUS       sequence-198818-        8337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory plasmid for PACE of MjTyrRS variants; negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8337)
  AUTHORS   Bryson DI, Fan C, Guo LT, Miller C, Soll D, Liu DR
  TITLE     Continuous directed evolution of aminoacyl-tRNA synthetases.
  JOURNAL   Nat Chem Biol. 2017 Oct 16. doi: 10.1038/nchembio.2474.
  PUBMED    29035361
REFERENCE   2  (bases 1 to 8337)
  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"; journalName: "Nat Chem 
            Biol. 2017 Oct 16. doi: 10.1038/nchembio.2474."
COMMENT     Sequence Label: pDB036d
FEATURES             Location/Qualifiers
     source          1..8337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             270..1544
                     /codon_start=1
                     /gene="III"
                     /product="pIII"
                     /label=M13 gene III
                     /note="minor coat protein"
                     /translation="MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTL
                     DRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGG
                     TKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQ
                     GALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
                     GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKM
                     ANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGS
                     NSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVF
                     AFLLYVATFMYVFSTFANILRNKES"
     primer_bind     complement(2227..2244)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2397..2483
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2575..2602
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5557..6348,6349..6417))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6180..6199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6418..6522)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6590..6608
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(6629..7579)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      7627..7849
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tgctgaggag
       61 acttagggac cctgatgaaa ttcgccactt gttagtgtaa ttcgctaact catcctggca
      121 tgttgctgtt gattcttcaa tcagatcttt ataaatcaaa aagataaaaa attggcacgc
      181 aaattgtatt aacagttcag caggacaatc ctgaacgcag aaatcaagag gacaacatta
      241 cctgcaggtg cagtaaggag gaaaaaaaaa tgaaaaaatt attattcgca attcctttag
      301 ttgttccttt ctattctcac tccgctgaaa ctgttgaaag ttgtttagca aaaccccata
      361 cagaaaattc atttactaac gtctggaaag acgacaaaac tttagatcgt tacgctaact
      421 atgagggctg tctgtggaat gctacaggcg ttgtagtttg tactggtgac gaaactcagt
      481 gttacggtac atgggttcct attgggcttg ctatccctga aaatgagggt ggtggctctg
      541 agggtggcgg ttctgagggt ggcggttctg agggtggcgg tactaaacct cctgagtacg
      601 gtgatacacc tattccgggc tatacttata tcaaccctct cgacggcact tatccgcctg
      661 gtactgagca aaaccccgct aatcctaatc cttctcttga ggagtctcag cctcttaata
      721 ctttcatgtt tcagaataat aggttccgaa ataggcaggg ggcattaact gtttatacgg
      781 gcactgttac tcaaggcact gaccccgtta aaacttatta ccagtacact cctgtatcat
      841 caaaagccat gtatgacgct tactggaacg gtaaattcag agactgcgct ttccattctg
      901 gctttaatga ggatccattc gtttgtgaat atcaaggcca atcgtctgac ctgcctcaac
      961 ctcctgtcaa tgctggcggc ggctctggtg gtggttctgg tggcggctct gagggtggtg
     1021 gctctgaggg tggcggttct gagggtggcg gctctgaggg aggcggttcc ggtggtggct
     1081 ctggttccgg tgattttgat tatgaaaaga tggcaaacgc taataagggg gctatgaccg
     1141 aaaatgccga tgaaaacgcg ctacagtctg acgctaaagg caaacttgat tctgtcgcta
     1201 ctgattacgg tgctgctatc gatggtttca ttggtgacgt ttccggcctt gctaatggta
     1261 atggtgctac tggtgatttt gctggctcta attcccaaat ggctcaagtc ggtgacggtg
     1321 ataattcacc tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg
     1381 aatgtcgccc ttttgtcttt ggcgctggta aaccttacga gttcagtatc gactgcgata
     1441 agatcaacct gttccgcggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg
     1501 tattttctac gtttgctaac atactgcgta ataaggagtc ttaatcttga gtaacggagc
     1561 caatgtacgc aaaaaacccc gcttcagcgg ggttttttcg ccaaaaaaaa ccccgccctg
     1621 tcaggggcgg ggtttttttt tagggcccta ctgactgtcc gtgtgcttct caaatgcctg
     1681 aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga tcagtgcacg
     1741 gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct attaagggat
     1801 tgacgagggc gtatctgcgc agtaagatgc gccccgcatt ccggcggtag ttcagcaggg
     1861 cagaacggcg gactctaaat ccgcatggca ggggttcaaa tcccctccgc cggaccaaat
     1921 tcgaaaagcc tgctcaacga gcaggctttt ttgcatgctc gagcagctca gggtcgaatt
     1981 tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt agcaaccagg
     2041 cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca
     2101 gtactgttgt aattcattaa gcattctgcc gacgttcatc ggatcccggg cccgtcgact
     2161 gcagaggcct gcatgcaagc ttggcgtaat cagtggctgt tttggcggat gagagaagat
     2221 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc
     2281 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaactccg
     2341 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc agtcatcaaa
     2401 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     2461 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc
     2521 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg
     2581 ccatcctgac ggatggcctt tttgcgtttc tacaaactcg ctgaggattc tagagcacag
     2641 ctaacaccac gtcgtcccta tctgctgccc taggtctatg agtggttgct ggataacttt
     2701 acgggcatgc ataaggctcg taggctatat tcagggagac cacaacggtt tccctctaca
     2761 aataattttg tttaactttt actagagtaa ggaggaaaaa tgaacacgat taacatcgct
     2821 aagaacgact tctaggacat cgaactggct gctatcccgt tcaacactct ggctgaccat
     2881 tacggtgagc gtttagctcg cgaacagttg gcccttgagc atgagtctta cgagatgggt
     2941 gaagcacgct tccgcaagat gtttgagcgt caacttaaag ctggtgaggt tgcggataac
     3001 gctgccgcca agcctctcat cactacccta ctccctaaga tgattgcacg catcaacgac
     3061 tggtttgagg aagtgaaagc taagcgcggc aagcgcccga cagccttcca gttcctgcaa
     3121 gaaatcaagc cggaagccgt agcgtacatc accattaaga ccactctggc ttgcctaacc
     3181 agtgctgaca atacaaccgt tcaggctgta gcaagcgcaa tcggtcgggc cattgaggac
     3241 gaggctcgct tcggtcgtat ccgtgacctt gaagctaagc acttcaagaa aaacgttgag
     3301 gaacaactca acaagcgcgt agggcacgtc tacaagaaag catttatgca agttgtcgag
     3361 gctgacatgc tctctaaggg tctactcggt ggcgaggcgt ggtcttagtg gcataaggaa
     3421 gactctattc atgtaggagt acgctgcatc gagatgctca ttgagtcaac cggaatggtt
     3481 agcttacacc gccaaaatgc tggcgtagta ggtcaagact ctgagactat cgaactcgca
     3541 cctgaatacg ctgaggctat cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg
     3601 ttccaacctt gcgtagttcc tcctaagccg tggactggca ttactggtgg tggctattgg
     3661 gctaacggtc gtcgtcctct ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc
     3721 tacgaagacg tttacatgcc tgaggtgtac aaagcgatta acattgcgca aaacaccgca
     3781 tggaaaatca acaagaaagt cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt
     3841 ccggtcgagg acatccctgc gattgagcgt gaagaactcc cgatgaaacc ggaagacatc
     3901 gacatgaatc ctgaggctct caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag
     3961 gacaaggctc gcaagtctcg ccgtatcagc cttgagttca tgcttgagca agccaataag
     4021 tttgctaacc ataaggccat ctggttccct tacaacatgg actggcgcgg tcgtgtttac
     4081 gctgtgtcaa tgttcaaccc gcaaggtaac gatatgacca aaggactgct tacgctggcg
     4141 aaaggtaaac caatcggtaa ggaaggttac tactggctga aaatccacgg tgcaaactgt
     4201 gcgggtgtcg ataaggttcc gttccctgag cgcatcaagt tcattgagga aaaccacgag
     4261 aacatcatgg cttgcgctaa gtctccactg gagaacactt ggtgggctga gcaagattct
     4321 ccgttctgct tccttgcgtt ctgctttgag tacgctgggg tacagcacca cggcctgagc
     4381 tataactgct cccttccgct ggcgtttgac gggtcttgct ctggcatcca gcacttctcc
     4441 gcgatgctcc gagatgaggt aggtggtcgc gcggttaact tgcttcctag tgaaaccgtt
     4501 caggacatct acgggattgt tgctaagaaa gtcaacgaga ttctacaagc agacgcaatc
     4561 aatgggaccg ataacgaagt agttaccgtg accgatgaga acactggtga aatctctgag
     4621 aaagtcaagc tgggcactaa ggcactggct ggtcaatggc tggcttacgg tgttactcgc
     4681 agtgtgacta agcgttcagt catgacgctg gcttacgggt ccaaagagtt cggcttccgt
     4741 caacaagtgc tggaagatac cattcagcca gctattgatt ccggcaaggg tctgatgttc
     4801 actcagccga atcaggctgc tggatacatg gctaagctga tttgggaatc tgtgagcgtg
     4861 acggtggtag ctgcggttga agcaatgaac tggcttaagt ctgctgctaa gctgctggct
     4921 gctgaggtca aagataagaa gactggagag attcttcgca agcgttgcgc tgtgcattgg
     4981 gtaactcctg atggtttccc tgtgtggcag gaatacaaga agcctattca gacgcgcttg
     5041 aacctgatgt tcctcggtca gttccgctta cagcctacca ttaacaccaa caaagatagc
     5101 gagattgatg cacacaaaca ggagtctggt atcgctccta actttgtaca cagccaagac
     5161 ggtagccacc ttcgtaagac tgtagtgtgg gcacacgaga agtacggaat cgaatctttt
     5221 gcactgattc acgactcctt cggtaccatt ccggctgacg ctgcgaacct gttcaaagca
     5281 gtgcgcgaaa ctatggttga cacatatgag tcttgtgatg tactggctga tttctacgac
     5341 cagttcgctg accagttgca cgagtctcaa ttggacaaaa tgccagcact tccggctaaa
     5401 ggtaacttga acctccgtga catcttagag tcggacttcg cgttcgcgta acttaattaa
     5461 cggcactgcg aaaaaacccc gccgaagcgg ggttttttgc gaaatataat gaccctcttg
     5521 ataacccaag agggcatttt ttaatgccca tggcgtttac caatgcttaa tcagtgaggc
     5581 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5641 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5701 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5761 cagaagcggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5821 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5881 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5941 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6001 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6061 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6121 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccagcgt caatacggga
     6181 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6241 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6301 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6361 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6421 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6481 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6541 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     6601 cacgaggccc ttaggccaaa taggccgttc agatccttcc gtatttagcc agtatgttct
     6661 ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatgcttac
     6721 tttgcatgtc actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag
     6781 catcgtgtag tgtttttctt agtccgttac gtaggtagga atctgatgta atggttgttg
     6841 gtattttgtc accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt
     6901 gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca
     6961 ccaatttcat attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt
     7021 taagcctttt aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa
     7081 ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact
     7141 cataaatcct catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta
     7201 tgaatttttt taactggaaa agataaggca atatctcttc actaaaaact aattctaatt
     7261 tttcgcttga gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag
     7321 gattcctgat ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat
     7381 aagcattttc cctactgatg ttcatcatct gagcgtattg gttataagtg aacgataccg
     7441 tccgttcttt ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa
     7501 ttagcttggt ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga
     7561 aaacaactaa ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat
     7621 tgagatgggc tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta
     7681 aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg
     7741 ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga
     7801 aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt
     7861 cagttccgca gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac
     7921 cttaaaaccc taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc
     7981 cttttgtctc cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct
     8041 gcgctcacgg ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt
     8101 catgcaagga aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt
     8161 atggcgggtc tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct
     8221 gattttccag tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc
     8281 acccagtaag gcagcggtat catcaacagg cttacccgtc ttactgtcaa gaggaca
//
