LOCUS       sequence-454421-        6273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory plasmid for negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  AUTHORS   Yang L, Zhang J, Andon JS, Li L, Wang T
  TITLE     Rapid discovery of cyclic peptide protein aggregation inhibitors by 
            continuous selection.
  JOURNAL   Nat Chem Biol. 2025 Jan 13. doi: 10.1038/s41589-024-01823-x.
  PUBMED    39806068
REFERENCE   2  (bases 1 to 6273)
  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. 2025 Jan 13. doi: 10.1038/s41589-024-01823-x."
COMMENT     Sequence Label: pTW357bN
FEATURES             Location/Qualifiers
     source          1..6273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     292..311
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     545..562
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    577..717
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(643..662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     803..825
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(819..1010)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             1311..1328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      complement(4763..4790)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4882..4968)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5121..5138
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(5184..5975)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPSQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5976..6047)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6076..6273,1..391)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
       61 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
      121 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
      181 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
      241 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      301 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      361 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      421 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      481 ggataaccgt attacagcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      541 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
      601 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
      661 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
      721 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtcttctccc ggcatccgct
      781 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
      841 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
      901 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
      961 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     1021 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     1081 acgatacggg ttactgatga tgaacaagag gacatccggt caaataaaac gaaaggctca
     1141 gtcgaaagac tgggcctttc gttttgctga ggagacttag ggaccctact aatacgtctc
     1201 actataggga gaaagacctg caggtgcagt aaaaaaggaa aaaaatgaaa aaattattat
     1261 tcgcaattcc tttagttgtt cctttctatt ctcactccgc tgaaactgtt catcaccatc
     1321 accatcacgc tgaaactgtt gaaagttgtt tagcaaaacc ccatacagaa aattcattta
     1381 ctaacgtctg gaaagacgac aaaactttag atcgttacgc taactatgag ggctgtctgt
     1441 ggaatgctac aggcgttgta gtttgtactg gtgacgaaac tcagtgttac ggtacatggg
     1501 ttcctattgg gcttgctatc cctgaaaatg agggtggtgg ctctgagggt ggcggttctg
     1561 agggtggcgg ttctgagggt ggcggtacta aacctcctga gtacggtgat acacctattc
     1621 cgggctatac ttatatcaac cctctcgacg gcacttatcc gcctggtact gagcaaaacc
     1681 ccgctaatcc taatccttct cttgaggagt ctcagcctct taatactttc atgtttcaga
     1741 ataataggtt ccgaaatagg cagggggcat taactgttta tacgggcact gttactcaag
     1801 gcactgaccc cgttaaaact tattaccagt acactcctgt atcatcaaaa gccatgtatg
     1861 acgcttactg gaacggtaaa ttcagagact gcgctttcca ttctggcttt aatgaggatc
     1921 cattcgtttg tgaatatcaa ggccaatcgt ctgacctgcc tcaacctcct gtcaatgctg
     1981 gcggcggctc tggtggtggt tctggtggcg gctctgaggg tggtggctct gagggtggcg
     2041 gttctgaggg tggcggctct gagggaggcg gttccggtgg tggctcttcc caaatggctc
     2101 aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat ttaccttccc
     2161 tccctcaatc ggttgaatgt cgcccttttg tctttggcgc tggtaaacct tacgagttca
     2221 gtatcgactg cgataagatc aacctgttcc gcggtgtctt tgcgtttctt ttatatgttg
     2281 ccacctttat gtatgtattt tctacgtttg ctaacatact gcgtaataag gagtcttaaa
     2341 cggagccaat gtacgcaaaa aaccccgctt cagcggggtt ttttcgccaa aaaaaacccc
     2401 gccctgtcag gggcggggtt tttttttagg gccctactga ctgttacgcg aacgcgaagt
     2461 ccgactctaa gatgtcacgg aggttcaagt tacctttagc cggaagtgct ggcattttgt
     2521 ccaattgaga ctcgtgcaac tggtcagcga actggtcgta gaaatcagcc agtacatcac
     2581 aagactcata tgtgtcaacc atagtttcgc gcactgcttt gaacaggttc gcagcgtcag
     2641 ccggaatggt accgaaggag tcgtgaatca gtgcaaaaga ttcgattccg tacttctcgt
     2701 gtgcccacac tacagtctta cgaaggtggc taccatcttg gctgtgtaca aagttaggag
     2761 cgataccaga ctcctgtttg tgtgcatcaa tctcgctatc tttgttggtg ttaatggtag
     2821 gctgtaagcg gaactgaccg aggaacatca ggttcaagcg cgtctgaata ggcttcttgt
     2881 attcctgcca cacagggaaa ccatcaggag ttacccaatg cacagcgcaa cgcttgcgaa
     2941 gaatctctcc agtcttctta tctttgacct cagcagccag cagcttagca gcagacttaa
     3001 gccagttcat tgcttcaacc gcagctacca ccgtcacgct cacagattcc caaatcagct
     3061 tagccatgta tccagcagcc tgattcggct gagtgaacat cagacccttg ccggaatcaa
     3121 tagctggctg aatggtatct tccagcactt gttgacggaa gccgaactct ttggacccgt
     3181 aagccagcgt catgactgaa ctcttagtca cactgcgagt aacaccgtaa gccagccatt
     3241 gaccagccag tgccttagtg cccagcttga ctttctcaga gatttcacca gtgttctcat
     3301 cggtcacggt aactacttcg ttatcggtcc cattgattgc gtctgcttgt agaatctcgt
     3361 tgactttctt agcaacaatc ccgtagatgt cctgaacggt ttcactagga agcaagttaa
     3421 ccgcgcgacc acctacctca tctcggagca tcgcggagaa gtgctggatg ccagagcaag
     3481 acccgtcaaa cgccagcgga agggagcagt tatagctcag gccgtggtgc tgtaccccag
     3541 cgtactcaaa gcagaacgca aggaagcaga acggagaatc ttgctcagcc caccaagtgt
     3601 tctccagtgg agacttagcg caagccatga tgttctcgtg gttttcctca atgaacttga
     3661 tgcgctcagg gaacggaacc ttatcgacac ccgcacagtt tgcaccgtgg attttcagcc
     3721 agtagtaacc ttccttaccg attggtttac ctttcgccag cgtaagcagt cctttggtca
     3781 tatcgttacc ttgcgggttg aacattgaca cagcgtaaac acgaccgcgc cagtccatgt
     3841 tgtaagggaa ccagatggcc ttatggttag caaacttatt ggcttgctca agcatgaact
     3901 caaggctgat acggcgagac ttgcgagcct tgtccttgcg gtacacagca gcggcagcac
     3961 gtttccacgc ggtgagagcc tcaggattca tgtcgatgtc ttccggtttc atcgggagtt
     4021 cttcacgctc aatcgcaggg atgtcctcga ccggacaatg cttccacttg gtgattacgt
     4081 tggcgaccgc taggactttc ttgttgattt tccatgcggt gttttgcgca atgttaatcg
     4141 ctttgtacac ctcaggcatg taaacgtctt cgtagcgcat cagtgctttc ttactgtgag
     4201 tacgcaccag cgccagagga cgacgaccgt tagcccaata gccaccacca gtaatgccag
     4261 tccacggctt aggaggaact acgcaaggtt ggaacatcgg agagatgcca gccagcgcac
     4321 ctgcacgggt tgcgatagcc tcagcgtatt caggtgcgag ttcgatagtc tcagagtctt
     4381 gacctactac gccagcattt tggcggtgta agctaaccat tccggttgac tcaatgagca
     4441 tctcgatgca gcgtactcct acatgaatag agtcttcctt atgccacgaa gaccacgcct
     4501 cgccaccgag tagaccctta gagagcatgt cagcctcgac aacttgcata aatgctttca
     4561 tttttttttc ctccttactc tagtaaaagt taaacaaaat tatttgtaga gggaaaccgt
     4621 tgtggtctcc ctgaatatag ataccgagcc ttatgcatgc ccgtaaagtt atccagcaac
     4681 cactcataga cctagggcag cagataggga cgacgtggtg ttagctgtgc tctagaatcc
     4741 tcagcgagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttaatttg
     4801 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac
     4861 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     4921 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt
     4981 tccctactct cgcatgggga gaccccacac taccatcggc gctacggcgt ttcacttctg
     5041 agttcggcat ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa ttctgtttta
     5101 tcagaccgct tctgcgttct gatttaatct gtatcaggct gaaaatcttc tctcatccgc
     5161 caaaacagcc atgcccatgg cgtttatttg ccgactacct tggtgatctc gcctttcacg
     5221 tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca
     5281 agataagcct gtctagcttc aagtatgacg ggctgatact gggacggcag gcgctccatt
     5341 gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct gtaccaaatg
     5401 cgggacaacg taagcactac atttcgctca tctccagccc agtcgggcgg cgagttccat
     5461 agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg atcaaagagt
     5521 tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt cagcaagata
     5581 gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc attgcgctgc
     5641 cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc
     5701 acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga agccgaagtt
     5761 tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac ggtcaccgta
     5821 accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga gccgtacaaa
     5881 tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac ctctgatagt
     5941 tgagtcgata cttcggcgat caccgcttcc ctcatactct tcctttttca atattattga
     6001 atcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6061 aggccaaata ggccgttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6121 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccac ctctttttcc
     6181 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     6241 gttaggccac cacttcaaga actctgtagc acc
//
